Dynamic Silk Coatings for Implantable Devices

ABSTRACT

Provided herein relates to implantable devices and systems with dynamic silk coatings. In some embodiments, the dynamic silk coatings can be formed in situ or in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. § 119(e) of the U.S.Provisional Application No. 61/477,484, filed Apr. 20, 2011, the contentof which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant no. P41EB002520 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

FIELD OF THE DISCLOSURE

Provided herein relates to silk-based implantable systems and devicesand methods of preparing the same. In some embodiments, the silk-basedimplantable systems and devices can be adapted for use asneuroprosthetic devices such as brain electrodes, shunts and/or spinalcord nerve guide wires.

BACKGROUND OF THE DISCLOSURE

Neuroprosthetic devices hold promise for future biomedical human-machineinteractions. Various applications of this technology are actively beingexplored, ranging from direct neural control of prosthetic limbs toaugmentation of sensory inputs. See, e.g., Andersen R A., et al.“Cognitive Neural Prosthetics.” Annu Rev Psychol. 2010; 61:169-C3;Venkatraman S, et al. “A System for Neural Recording and Closed-LoopIntracortical Microstimulation in Awake Rodents.” IEEE Transactions onBiomedical Engineering. 2009 January; 56(1):15-22. Indeed, firstgeneration neuroprosthetics, such as cochlear implants, have alreadybeen used in the clinic (Fayad G and Elmiyeh B. “Cochlear Implant.” In:Hakim N S, editor. Artificial Organs. London: Springer London; 2009; p.133-6). Despite this success, the long-term reliability of chroniccortical electrode implants remains a major obstacle to the widespreadadaptation of neuroprosthetic technologies to the clinic, with manypenetrating arrays losing the ability to record neurons after just weeksor months.

This lack of chronic reliability is often attributed to gliosis, theinflammatory response in the central nervous system. Gliosis ischaracterized by the formation of a glial scar around the implantedelectrode in an attempt by the body to wall off the injury site fromhealthy tissue. A dense accumulation of microglia, macrophages, andreactive astrocytes produce extracellular matrix molecules that inhibitaxonal growth within the scar. This encapsulation effectively increasesthe electrical impedance at the recording site, while also acting as aphysical barrier between the electrode and the targeted neuronalpopulations. As the glial scar grows, the electrode becomes incapable ofrecording extracellular action potentials.

Bioelectrodes for neural recording and neurostimulation are an integralcomponent of a number of neuroprosthetic devices, including commerciallyavailable cochlear implants and developmental devices, such as bioniceyes and brain-machine interfaces. Deep brain stimulation (DBS) is anestablished therapy for the treatment of Parkinson's disease (PD) andshows promise for the treatment of several other disorders, where it isessential to have a spatially precise contact between the electrode andtissue. Current rigid metal-based electrodes can acquire signals over afew days to weeks. However, continuing to extract signals with a highfidelity over long periods of time remains a major challenge. Presently,issues regarding electrode fracture and signal drop-out plaguemetal-based micro-electrodes for long-term use. Rigid metal needles donot comply mechanically with brain tissue, shifting during normal headmovement, resulting in electrode misplacement from the target neuraltissue area and electrode breaks.

The large mismatch between the elastic modulus of brain tissue andconventional silicon-based electrode shanks has been implicated as asignificant factor contributing to chronic gliosis. Finite elementanalysis techniques have been used to model the large stressconcentrations and “micromotion” effect that occur at thebrain-electrode interface in response to indwelling, inelastic materials(Subbaroyan J. et al., “A finite-element model of the mechanical effectsof implantable microelectrodes in the cerebral cortex.” Journal ofNeural Engineering. 2005 Dec. 1; 2(4):103-13; Lee H, Bellamkonda R V. etal., “Biomechanical analysis of silicon microelectrode-induced strain inthe brain.” Journal of Neural Engineering. 2005 Dec. 1; 2(4):81-9).Electrode micromotion resulting from a mismatch in stiffness between theelectrode and the brain tissue leading to chronic inflammation andassociated increased glial activation, and movement of the electrodefrom the target, resulting in inconsistent readings. Mechanical stresshas been shown to stimulate astrocyte reactivity and neuronal death invitro (Cullen D K et al., “Strain rate-dependent induction of reactiveastrogliosis and cell death in three-dimensional neuronal-astrocyticco-cultures.” Brain Research. 2007 Jul. 16; 1158(0):103-15), and in vivostudies have reported increased scarring around electrodes tethered tothe skull, which are mechanically less compliant than free-floatingprobes (Thelin J, et al. “Implant Size and Fixation Mode StronglyInfluence Tissue Reactions in the CNS.” PLoS ONE. 2011 Jan. 26;6(1):e16267; Biran R. et al., “The brain tissue response to implantedsilicon microelectrode arrays is increased when the device is tetheredto the skull.” J Biomed Mater Res A. 2007 July; 82(1):169-78). A recentin vivo report compared the tissue response to stiff and compliantprobes with the same surface chemistry, finding the compliant probesreduced glial scar intensity, with a greater density of nearby neuronscompared to the stiff shanks at 4 weeks (Harris J P, et al.“Mechanically adaptive intracortical implants improve the proximity ofneuronal cell bodies. Journal of Neural Engineering.” 2011 Oct. 1;8:066011).

One approach to minimize electrode micromotion and associated glialscarring, and/or to provide strain relief at the electrode-issueinterface is to use flexible, thin-film probes fabricated from polymerssuch as polyimide or parylene. See, e.g., Rousche P J, et al., “Flexiblepolyimide-based intracortical electrode arrays with bioactivecapability.” Biomedical Engineering, IEEE Transactions on. 2001;48(3):361-71; Mercanzini A, et al. “Demonstration of cortical recordingusing novel flexible polymer neural probes.” Sensors and Actuators A:Physical. 2008 May 2; 143(1):90-6; Hess A E, et al. “Development of astimuli-responsive polymer nanocomposite toward biologically optimized,MEMS-based neural probes.” J. Micromech. Microeng. 2011 May;21(5):054009; Kato Y. et al., “Preliminary Study of MultichannelFlexible Neural Probes Coated with Hybrid Biodegradable Polymer.” In:Engineering in Medicine and Biology Society, 2006. EMBS '06. 28th AnnualInternational Conference of the IEEE. 2006. p. 660-3; Takeuchi S. et al.“Parylene flexible neural probes integrated with microfluidic channels.”Lab Chip. 2005 May; 5(5):519-23; Suzuki T, et al., “A 3D flexibleparylene probe array for multichannel neural recording.” In: NeuralEngineering, 2003. Conference Proceedings. First International IEEE EMBSConference. 2003. p. 154-6; Wester B A, et al., “Development andcharacterization of in vivo flexible electrodes compatible with largetissue displacements.” J. Neural Eng. 2009 April; 6(2):024002; andSeymour J P and Kipke D R. “Neural probe design for reduced tissueencapsulation in CNS.” Biomaterials. 2007 September; 28(25):3594-607.Such flexible probes introduce an additional challenge, however, asthese devices can be incapable of penetrating the pia to achieve preciseinsertion into the brain without buckling.

Further, the neural recording interface remains another major challenge.Erosion of connections between the abiotic and biotic niches results inloss of function of electrodes and implants. This is an endemic problemin biology and one that has resisted traditional approaches forresolution. As a result, implants for recording electronics haverelatively short lifetimes, requiring constant replacement, leading toassociated failure modes due to the device itself or repeated surgicalintervention that damages surrounding tissue.

Since most electrode implants remain relatively rigid and damaging uponinsertion, the interface between current systems and the dynamic natureof biological systems tend to be a major source of problems. The dynamicissues include the biochemistry at the site (cell signaling factors, ECMdeposition), cell dynamics at the site (e.g., astroglial and other nervecells that respond to and with the implants), and stress shielding(e.g., mechanical mismatch between electrodes and soft brain tissue,leading to failure modes, fibrous encapsulation and relatedcomplications and failures to maintain a stable interface). Further, arelated underlying problem includes the limited ability to establish aconformal and tight interface, due to the mechanical mismatch, betweenthe existing materials and the soft and convoluted brain andneurological tissues. Therefore, there is a need to develop technologiesand/or devices that can overcome one or more of the above limitations.

SUMMARY

The neural recoding interface remains a major challenge. While flexibleelectrodes are desired to be used for their better compliance with abrain tissue, these flexible electrodes are not stiff enough topenetrate into a brain tissue. Further, erosion of connections betweenthe abiotic and biotic niches results in a loss of function ofelectrodes and implants. As a result, implants (e.g., for recodingelectronics) have relatively short lifetimes, thus requiring constantreplacement and leading to associated failure modes due to the deviceitself or repeated surgical intervention that damages surroundingtissue. Thus, there is a need for developing implantable devices and/orsystems with sufficient mechanical stiffness for insertion into a braintissue without compromising the mechanical compliance with a braintissue after insertion. Provided herein generally relates to dynamicsilk coatings for implantable devices, wherein the silk coating can beapplied onto an implantable flexible device before implantation, as ameans to provide the flexible device with mechanical strength, whichwill soften upon implantation, and/or the silk coating can be renewablyformed in situ on a surface (e.g., a conducting surface) of theimplantable device post-implantation, e.g., to minimize bio-fouling.

Accordingly, one aspect provided herein relates to a silk-basedimplantable system comprising an electrical component, wherein at leasta portion of the electrical component is in contact with a silk matrix,the silk matrix providing the electrical component with sufficientstiffness to penetrate a target tissue and becoming compliant upon thepenetration.

In some embodiments, the electrical component can be at least part of apre-formed or a conventional implantable device that would generallydeform during the penetration in the absence of the silk matrix.Accordingly, in such embodiments, coating the preformed or conventionalimplantable device can provide it with mechanical stiffness sufficientto penetrate into a target tissue (e.g., without deformation such asbuckling or bending). Upon penetration, the silk coating can becomesofter, thus allowing the implantable device to be mechanicallycompliant with the target tissue.

A preformed or conventional implantable device can be an implantabledevice for any part of the body in a subject including, but are notlimited to, subcutaneous, intramuscular, intraperitoneal, cardiac,pulmonary, and neural tissue. In some embodiments. The pre-formed orconventional implantable device can be a neuroprosthetic device.Exemplary neuroprosthetic device can include, but are not limited to, abrain penetrating electrode, a shunt, a nerve guide, a cochlear implant,and a microelectrode array.

While the electrical component can be an integral part of a pre-formedor conventional implantable device, the electrical component can also bedirectly or indirectly patterned on the silk matrix to form thesilk-based implantable device.

The electrical component can be formed from any conventional materialscommonly used for electronics in an implantable device, e.g., withoutlimitations, silicon, gold, platinum, titanium, and any combinationsthereof. Alternatively, the electrical component can includebiodegradable electronics, e.g., made from biodegradable or erodibleorganic semiconductors such as melanins and carotenoids. In oneembodiment, the electrical component can include a silk-based electrodeas described herein.

In accordance with various aspects described herein, the silk matrix isdynamic with respect to its changes in the mechanical property before orduring and after penetration (or between a dry state and a hydratedstate). Before penetration and during at least part of the penetrationprocess, the silk matrix is stiff enough to enable the implantabledevice to penetrate into a target tissue (e.g., without deformation suchas buckling or bending). In some embodiments, the silk matrix canincrease a buckling force of the electrical component or the implantabledevice by at least about 2-fold, at least about 5-fold, at least about10-fold or more, as compared to the absence of the silk matrix. In someembodiments, the silk matrix can increase a buckling force of theelectrical component or the implantable device by at least about 1 orderof magnitude (e.g., about 10-fold or more), about 2 orders of magnitude(e.g., about 100-fold or more), about 3 orders of magnitude (e.g., about1000-fold or more) or more, as compared to the absence of the silkmatrix.

Upon penetration into the target tissue, the silk matrix in contact withthe electrical component can become compliant upon the penetration,e.g., by hydration of the silk matrix. Such mechanical compliance of thesilk matrix with surrounding tissue upon penetration can provideconformal contact between the electrical component and a surface of thetarget tissue.

In some embodiments, at least one side of the electrical component canbe coated with the silk matrix. Thus, at least part of the electoralcomponent can still be exposed to surrounding tissue, e.g., fordetecting a signal. Without wishing to be bound, the electricalcomponent can also be encapsulated in the silk matrix. In suchembodiments, the silk matrix can be modified or doped to become aconductive material.

In some embodiments, the silk matrix can reduce scar formation (e.g.,gliosis) around the electrical component or the implantable device by atleast about 10%, as compared to the absence of the silk matrix.

Silk matrix can stabilize an active agent including a therapeutic agentfor an extended release in vivo. Accordingly, in some embodiments, thesilk matrix can comprise an active agent including, but not limited to,a therapeutic agent. The active agent (including a therapeutic agent)can include, e.g., but are not limited to, an agent that promotes tissuegrowth, controls inflammation, and/or reduces scar formation around theimplanted system or device. In one embodiment, the active agent caninclude a gliosis-modulating agent.

Depending on applications of the implantable device and/or mechanicalproperty of implantation sites, the silk matrix can have a thickness ofabout 1 μm to about 1000 μm. The total thickness of the silk matrix canbe resulted from a single layer or a plurality of silk layers, each ofwhich can have the same or different thickness and optionally the sameor different active agent.

Applications of the silk-based implantable systems are also providedherein. For example, a method of inserting a flexible or softimplantable device into a target tissue comprises providing a silk-basedimplantable system described herein, wherein the silk-based implantablesystem comprises the flexible or soft implantable device at leastpartially coated with a silk matrix in its dry-state. In someembodiments, the silk-based implantable systems can be used to reducescar formation (e.g., gliosis) around the implantable device (e.g.,neuroprosthetic device) implanted in a tissue (e.g., a brain tissue). Insome embodiments, the silk-based implantable systems can be used toimprove long-term functionality of an implantable device (e.g., aneuroprosthetic device) implanted in a tissue (e.g., a brain tissue).

Another aspect described herein relates to a silk-based implantabledevice comprising a silk body with at least one electrically-conductingcomponent. In some embodiments, the silk-based implantable device can beconstructed to be capable of renewably forming a silk coating on asurface of the implantable device. Such silk-based implantable devicecan minimize biofouling, thus increasing the reliability and lifetime ofthe implantable device in vivo.

For example, in some embodiments, the silk body can include a silkreservoir of any shape with at least one electrically-conductingcomponent formed on a least a portion of a surface of the silk reservoir(e.g., the surface that is in contact with a fluid upon implantation).In some embodiments where the silk-based implantable device are adaptedfor use as an electrode, the silk reservoir can be a silk tube with atleast one electrically-conducting component formed on at least a portionof a lateral surface of the silk tube. The silk reservoir or silk tubecan be filled with a silk solution.

In alternative embodiments, the silk body can comprise a first silklayer and a second silk layer, wherein at least one of the first and thesecond silk layer can comprise at least one electrically-conductingcomponent formed on at least a portion of a surface of the silk layer.In some embodiments, between the first and the second silk layers caninclude solid-state silk that can be solubilized to form a silk solutionupon contact with a fluid, e.g., silk particles (including lyophilizedsilk particles), silk powder (including lyophilized silk powder), or asilk film.

To allow the silk solution to form a coating on a surface of theelectrically-conducting component, in some embodiments, theelectrically-conducting component can include one or more through holessuch that the silk solution present in the silk reservoir (e.g., silktube) between the silk layers can be discharged onto the surface of theelectrically-conducting component to be coated. The solution can bedischarged onto the surface of the electrically-conducting component tobe coated by any methods known in the art, including, but not limitedto, diffusion, an implantable pump, and/or an external pump. The silksolution discharged onto the surface of the electrically-conductingcomponent can form a gel-like coating upon application of a firstvoltage through the electrically-conducting component. In someembodiments, the gel-like coating can be removed, e.g., by transformingthe gel-like coating to a solution upon application of a second voltagewith a polarity opposite to the first voltage.

The electrical conducting component can include any material that iscommonly used as electronics for implantable devices, and/or anelectrically-conductive material. In some embodiments, the electricalconducting component can include a metal such as a transition metal(e.g., silicon), a noble metal (e.g., gold), or a combination thereof. I

In some embodiments, the electrical conducting component can include abiodegradable component that can conduct electricity such asbiodegradable organic semiconductors (e.g., melanins and/orcarotenoids).

In some embodiments, the electrical conducting component can includesilk modified to conduct electricity. For example, the silk can befunctionalized by modifying a tyrosine of the silk protein to a sulfategroup followed by polymerization of the modified tyrosine with aconducting polymer. Alternatively, the silk can be doped with aconductive material including, but not limited to, gold nanoparticles,carbon nanotubes, graphene, and a conducting polymer. Non-limitingexamples of a conducting polymer can include polyethylenedioxythiophene(PEDOT), polypyrrole-based conductive polymer, copolymers of thiophenesand polypyrroles, copolymers of poly-lactide and polyaniline, or anycombinations thereof.

In some embodiments, the silk-based implantable device can be adaptedfor use as an implantable brain penetrating electrode. For example, thesilk body can be in a form of a silk tube with a diameter of less than 2mm. In some embodiments, the silk-based electrode can have a tensilestrength of at least about 2 MPa when the silk body is in a dry state.In some embodiments, the silk-based electrode can have a shear modulusof less than about 200 kPa upon contact of the silk body with a fluid(e.g., interstitial fluid and/or body fluid such as cerebrospinalfluid).

In some embodiments, the silk body can comprise an active agentincluding a therapeutic agent.

Methods for regenerating a silk coating on a surface of a device arealso provided herein. In some embodiments, the method comprisesproviding the silk-based device described herein, wherein the silksolution discharged onto the surface of the electrically-conductingcomponent can form a gel coating upon application of a first voltagethrough the electrically-conducting component, and can optionally turnto a solution upon application of a second voltage with a polarityopposite to the first voltage.

The capability of renewing or regenerating the silk coating on a surfaceof a device can reduce biofouling. Accordingly, a method of reducingbiofouling of a device is provided herein. The method comprisesproviding the silk-based device described herein, wherein the silksolution discharged onto the surface of the electrically-conductingcomponent forms a gel coating upon application of a first voltagethrough the electrically-conducting component, and can optionally turnto a solution upon application of a second voltage with a polarityopposite to the first voltage. The first voltage and/or the secondvoltage can be applied to the electrically-conducting component at anypotential, provided that the voltage potential is high enough for silkgelation. In some embodiments, the first voltage and the second voltagecan be at least about 1.2V. In other embodiments, the first voltage andthe second voltage can be about 5 V to about 50 V.

In some embodiments, the device is placed in vivo or in situ. Thus,methods described herein can be carried out in vivo or in situ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1H are representative images of glial fibrillary acid protein(GFAP) or chondroitin sulfate (CS) staining around different treatedsteel microwires in an in vitro glial scarring model. FIGS. 1A and 1Bare representative images of GFAP staining around uncoated andsilk-coated steel microwires, respectively. FIGS. 1C and 1D arerepresentative images of CS staining around uncoated and silk-coatedsteel microwires, respectively. FIGS. 1E and 1F are representativeimages of GFAP staining around a steel microwire dipped in 25 mg/mLcytarabine (Ara-C) in H₂O and a silk solution containing 25 mg/mL Ara-C,respectively. FIGS. 1G and 1H are representative images of CS stainingaround a steel microwire dipped in 25 mg/mL cytarabine (Ara-C) in H₂Oand a silk solution containing 25 mg/mL Ara-C, respectively. Microwirediameter=50 μm.

FIGS. 2A and 2B show average scar index values for in vitro glial scarcultures stained for GFAP and CS, respectively. * p<0.05, ** p<0.005,*** p<0.0005.

FIG. 3 is an image showing an exemplary flexible electrode fabricated byencapsulating gold traces in parylene C. Additional details of theflexible electrode can be found in Metallo C. et al., “Flexibleparylene-based microelectrode arrays for high resolution EMG recordingsin freely-moving small animals.” J Neurosci Methods. (2011) 15;195(2):176-84.

FIG. 4 is an image showing exemplary electrical components such as wirespartially coated with silk fibroin.

FIGS. 5A-5C shows penetration capability of uncoated and silk-coatedwires through a Parafilm membrane. FIG. 5A shows that uncoated wirecannot penetrate Parafilm membrane. FIG. 5B shows that silk-coated wirepenetrates Parafilm without buckling.

FIG. 5C shows that after 30 seconds hydration, the silk-coated wirebecomes compliant, and cannot penetrate Parafilm.

FIGS. 6A-6C show exemplary methods for coating flexible substrates insilk. FIG. 6A shows a dip-coating method. FIG. 6B shows a single-stepcasting. FIG. 6C shows a two-step casting.

FIGS. 7A and 7B show side view of uncoated (˜60 um thickness) andsilk-coated (˜120 um thickness) parylene strips, respectively. Coatingwas applied via two-step casting. Bar=200 micrometers.

FIG. 8 shows top view of parylene coated in silk by two-step casting.Bar=500 micrometers.

FIG. 9 shows an exemplary flexible electrode (left) coated in silk(right) using the two-step casting method. The shaft of the electrode isfully encapsulated in silk, while the top connector area has silk onlyon the back.

FIGS. 10A-10C show force curves for five replicates of uncoated parylene(FIG. 10A), silk-coated parylene (FIG. 10B) and silk-coated paryleneallowed to hydrate for 30 minutes in 37° C. PBS (FIG. 10C).

FIG. 11 show average buckling forces (in log scale) for differenttreated parylene (uncoated parylene, silk-coated parylene, andsilk-coated parylene after hydration).

FIGS. 12A-12B are schematic diagrams of a silk-based electrode accordingto different embodiments described herein. FIG. 12A is a schematicdiagram of a silk electrode with a reservoir for silk solution. FIG. 12Bis a schematic diagram of a silk electrode designed for regenerating asilk coating using lyophilized silk powder.

FIGS. 13A-13D are images of implantable silk shanks and silk-coatedelectrodes. FIG. 13A is an image showing diameter measurement of aneedle-like silk matrix. FIG. 13B is an image of a needle-like silkmatrix with a sharpened tip. FIG. 13C is an image showing diametermeasurement of silk implantable electrodes with different diameters.FIG. 13D is an image showing a silk implantable electrode as a foam-likesilk construct with embedded motor wire conductor (top) and sharpenedfor enhanced puncturing ability (bottom).

FIGS. 14A-14B show a silk electrogelation (e-gel) process applied to an8 wt % silk aqueous solution with 25 VDC using mechanical pencil leadelectrodes. FIG. 14A shows that over 3 minutes the gel forms around thepositive electrode with gas evolution at both electrodes. FIG. 14B showsthat gelation is reversed with the application of reversed polarity DCvoltage.

FIGS. 15A-15D show mechanical characteristics data for electrogelationof silk solutions as determined by dynamic oscillatory shear rheology.FIGS. 15A and 15B show dynamic shear frequency and strain sweeps,respectively, collected from silk solutions prior and post processing.In both FIGS. 15A and 15B, 25 VDC voltage was applied to 2 mL of 8.4 wt% silk solution for 10 min using platinum wires for e-gel formation. Forthe pH-gel, 8.4 wt % silk solution (pH 6.5) was treated by a strong acid(1 M HCl) to adjust the final proton concentration due to the acid to0.01 M (pH ˜4.4). Solid symbols in FIG. 15B show the strain response ofe-gels and silk solutions after application of high amplitude shear.FIG. 15C show engineering stress-strain curves obtained from the silksolution and e-gel by transient tensile testing of adhesion on thestainless steel surfaces of the DMA. Inset shows the initial linearregion of the engineering stress-strain curves. FIG. 15D is a photographof the e-gel at intermediate strains during transient testing displayingadhesive properties.

FIG. 16 is a set of images showing exemplary gel spun silk tubesindicating the versatility in engineering control of winding patterns(top left), tube wall porosity (top right), and loading of therapeuticsor growth factors (bottom panel) in gradient and layered locations(shown with dyes for easy tracking).

FIGS. 17A-17D show adenosine releasing silk implants for local epilepticseizure control. FIG. 17A is a schematic diagram of silk microspheresimbedded in silk sponge rods (0.8 mm diameter, 4 mm length) coated withadditional drug-loaded films and implanted at site of epileptic focus ina kindling rodent model. FIG. 17B shows a histological section of silkimplant after one month in the brain. FIG. 17C is a plot showing invitro release kinetics of adenosine from the silk implants. FIG. 17D isa plot showing seizure suppression (indicated by an arrow) through 13stimulations/6 days in vivo.

FIGS. 18A-18C are schematic illustration and images corresponding tosteps for fabricating conformal silk-supported PI electrode arrays. FIG.18A shows casting and drying of silk fibroin solution on a temporarysubstrate of PDMS; 5-15 μm thick silk film after drying for 12 hours atroom temperature. FIG. 18B shows steps for fabricating the electrodearrays, transfer printing them onto silk, and connecting to anisotropicconductive film (ACF) cable. FIG. 18C shows schematic illustration ofclinical usage of a representative device in an ultrathin mesh geometrywith dissolvable silk support.

FIGS. 19A-19D show neural electrode arrays of varying thickness onsimulated brain models to illustrate flexibility. FIG. 19A showsschematic illustration of trends in thickness and structure that improveconformal contact. FIG. 19B shows a series of pictures illustrating howthe thickness of the electrode array contributes to conformal contact ona brain model. FIG. 19C show magnified view of the pictures from FIG.19B. FIG. 19D is an image of an electrode array with a mesh design ondissolvable silk substrate. Arrows indicate struts in the mesh that helpto stabilize the Au interconnects after dissolution of the silk. Theinset illustrates the high degree of conformal contact that can beachieved on the brain model once the silk substrate has been dissolved.

FIG. 20 is a plot of silk processing relationships to degradation rateshowing programmable degradation lifetime. Further, silk is an FDAapproved biomaterial for use in a biomedical polymer system due tocapability of processing the silk protein to avoid inflammation whilepreserving biodegradability.

FIGS. 21A and 21B show exemplary stabilization of enzymes in silk filmsby entrainment over time (months) from 4 to 37° C., indicating that silkcan stabilize a gliosis-modulating agent for glial scarring control.FIG. 21A indicates stabilization data for lipase in silk films. FIG. 21Bindicates stabilization data for peroxidase in silk films.

DETAILED DESCRIPTION

While flexible electrodes are desirable for implantation due to theirbetter compliance with a brain tissue, these flexible electrodes aregenerally not stiff enough to penetrate into a brain tissue. Further,implants (e.g., for recoding electronics) have relatively shortlifetimes, partly due to erosion of connections between the abiotic andbiotic niches. Thus, the reliability of chronic flexible electrodes(e.g., chronic brain penetrating electrodes for neural recording) needsto be improved in order for the technology to be viable in clinicalapplications. One approach is to improve implantable devices and/orsystems with sufficient mechanical stiffness for insertion into a braintissue without compromising the mechanical compliance of a flexibleimplant with a brain tissue after insertion, and to reduce theinflammatory response at the probe-tissue interface. Provided hereingenerally relates to dynamic silk coatings for implantable devices,wherein the silk coating can be applied onto an implantable flexibledevice before implantation, as a means to provide the flexible devicewith mechanical strength, which will soften upon implantation, and/orthe silk coating can be renewably formed in situ on a surface (e.g., aconducting surface) of the implantable device post-implantation, e.g.,to minimize bio-fouling.

Silk-Based Implantable Systems and Methods of Making the Same

The next generation of neuroprosthetic devices can rely on chronicallyimplanted arrays of small electrodes targeting specific neuronalclusters within the brain. The existing cortical electrodes generallyconsist of stiff spikes of metal or silicon, and have been showninadequate for chronic neural sensing due to both a slow degradation ofsignal over time as well as inconsistent targeting of neurons. Two majorfactors that can contribute to the lack of chronic electrode reliabilityinclude (1) glial scar formation around the electrode leading to signaldegradation, e.g., from increased impedance and/or neuronalre-arrangement and death; and (2) electrode micromotion resulting fromthe mismatch in stiffness between the electrode and the brain leadingto, e.g., chronic inflammation and thus increased glial activation,and/or movement of the electrode from the target, thus giving consistentreadings. See, e.g., Polikov V. S. et al., “Response of brain tissue tochronically implanted neural electrodes,” Journal of NeuroscienceMethods, vol. 148, no. 1, pp. 1-18, October 2005; and Leach J. B. etal., “Bridging the Divide between Neuroprosthetic Design, TissueEngineering and Neurobiology,” Frontiers in Neuroengineering, vol. 2, p.18, 2010.

To address the issue of electrode micromotion, flexible electrodes havebeen suggested as an alternative to stiff designs. However, onechallenge with such flexible electrodes is achieving insertion into thebrain without buckling. Various insertion techniques have been reported,including placement with a removable needle (D. O'Brien, T. Nichols, andM. Allen, “Flexible microelectrode arrays with integrated insertiondevices,” in Micro Electro Mechanical Systems, 2001. MEMS 2001. The 14thIEEE International Conference on, pp. 216-219, 2001), or encapsulationwith a stiff, biodegradable substrate such as PEG (T. Suzuki, K.Mabuchi, and S. Takeuchi, “A 3D flexible parylene probe array formultichannel neural recording,” in Neural Engineering, 2003. ConferenceProceedings. First International IEEE EMBS Conference on, pp. 154-156,2003; and S. Takeuchi, D. Ziegler, Y. Yoshida, K. Mabuchi, and T.Suzuki, “Parylene flexible neural probes integrated with microfluidicchannels,” Lab on a Chip, vol. 5, no. 5, pp. 519-523, May 2005) orgelatin (G. Lind, C. E. Linsmeier, J. Thelin, and J. Schouenborg,“Gelatine-embedded electrodes—a novel biocompatible vehicle allowingimplantation of highly flexible microelectrodes,” Journal of NeuralEngineering, vol. 7, no. 4, p. 046005, August 2010). Using suchbiodegradable substrate can provide the ability to deliver bioactivecompounds such as anti-inflammatory agents to reduce glial scarring, orneuronal stimulants such as NGF. See, e.g., Y. Kato, I. Saito, T.Hoshino, T. Suzuki, and K. Mabuchi, “Preliminary Study of MultichannelFlexible Neural Probes Coated with Hybrid Biodegradable Polymer,” inEngineering in Medicine and Biology Society, 2006. EMBS '06. 28th AnnualInternational Conference of the IEEE, pp. 660-663, 2006.

However, such combinatorial approach, which allows for a stiff toflexible transition in mechanics, as well as long-term drug release, forincreasing the long-term reliability of penetrating electrodes (e.g.,reducing local glial scarring while promoting neuron survival) requiresthe development of more advanced electrode substrates. Many of thematerials that have been reported to provide dynamic mechanicalproperties are not well suited for sustained local release oftherapeutics. For example, stiffening materials prepared via evaporationof chemical solvents (9,21) can preclude the incorporation and releaseof sensitive molecules such as enzymes and growth factors. In addition,most of the previously-reported coating materials can dissolve withinminutes after hydration (14,15,18-20), making it impossible to achievesustained drug release over days or weeks. Encapsulation ofbiodegradable, drug-loaded micro or nano spheres within the dissolvablestiffening materials has been previously reported as a means to achievelonger release (13,25). However, the localization of the spheres at theimplant-tissue interface cannot be likely sustained after theencapsulating material dissolves.

The inventors have shown that, in particular embodiments, silk fibroincan provide an improved material platform for fabricating implantablesystems (e.g., implantable electrical systems such as chronic brainpenetrating electrodes) that are mechanically dynamic (e.g., forplacement of the implantable system to a target tissue) and capable ofextended local drug release (e.g., of one or more gliosis-modulatingagents). By way of example only, the silk can initially providemechanical stability for penetration of a brain penetrating electrode,while becoming flexible after hydrating in the aqueous environment ofthe brain after insertion. In some embodiments, the silk can degradeover time, leaving the flexible electrodes in place. Further, thedynamic silk coatings of an implantable device can reduce inflammatoryresponse and thus scar formation (e.g., gliosis) around the implantabledevice.

Accordingly, one aspect described herein relates to a silk-basedimplantable system comprising an electrical component, wherein at leasta portion of the electrical component is in contact with a silk matrix.The properties of the silk matrix can be tailored to provide theelectrical component with sufficient stiffness to penetrate a targettissue and become compliant or flexible upon the penetration. In someembodiments, such dynamic properties can be achieved by a change inhydration (e.g., water content of the silk matrix). In otherembodiments, the silk can degrade to allow flexibility. If degradationis required, delivery of an active agent over an extended period can bereconciled, e.g., encapsulating the active agent in another matrix(e.g., silk particles or other polymeric particles) which is thenembedded into the silk matrix.

As used herein, the term “penetration” or “penetrate” is generally meantby a silk-based implantable system or device described herein passingthrough at least one barrier (e.g., one or more membranes encapsulatinga tissue such as brain dura and/or pia mater) and reaching a certaindepth into the tissue. For example, the silk-based implantable system ordevice described herein can pass through at least one barrier (e.g.,meninges including dura mater, arachnoid mater, and pia mater) and reachat least about 50 μm into the tissue, including at least about 100 μm,at least about 200 μm, at least about 300 μm, at least about 400 μm, atleast about 500 μm, at least about 1 mm, at least about 2 mm, at leastabout 3 mm, at least about 4 mm, at least about 5 mm or deeper, into thetissue. In some embodiments, the silk-based implantable system or devicedescribed herein can pass through at least one barrier (e.g., meningesincluding dura mater, arachnoid mater, and pia mater) and reach at leastabout 5 mm, at least about 1 cm, at least about 2 cm, at least about 3cm, at least about 4 cm, at least about 5 cm, at least about 6 cm, atleast about 7 cm, at least about 8 cm, at least about 9 cm, at leastabout 10 cm or deeper, into the tissue. The terms “insert” and“penetrate” are used interchangeably herein.

As used interchangeably herein, the phrases “sufficient stiffness topenetrate a target tissue” and “stiff enough for insertion” refer to asilk-based implantable system and/or device described herein, in its drystate, having a Young's modulus at least comparable to or greater thanthat of a target tissue (e.g., a brain tissue) to be penetrated and/or abarrier (e.g., a protective tissue layer such as meninges) surroundingthe target tissue. In some embodiments, the phrases can refer to thesilk-based implantable system and/or device described herein, in its drystate, having a Young's modulus, which is at least about 5% greater(including at least about 10% greater, at least about 20% greater, atleast about 30% greater, at least about 40% greater, at least about 50%greater, at least about 60% greater, at least about 70% greater, atleast about 80% greater, at least about 90% or higher) than that of atarget tissue (e.g., a brain tissue) to be penetrated and/or a barrier(e.g., a protective tissue layer such as meninges) surrounding thereof.For example, for penetration into a brain tissue, the silk-basedimplantable system and/or device can have a Young's modulus of more than1 MPa, including, e.g., 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa,9 MPa, 10 MPa, 15 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa,80 MPa, 90 MPa, 100 MPa, or higher, when it is in a dry-state. In someembodiments, the silk-based implantable system and/or device can have aYoung's modulus of more than 100 MPa, 250 MPa, 250 MPa, 500 MPa, 1000MPa, 2500 MPa, 5000 MPa, 7500 MPa, 10,000 MPa, or higher, when it is ina dry-state. As used herein, the term “dry-state” refers to a silkmatrix being hydrated (e.g., water content) for no more than 30%, nomore than 20%, no more than 10%, no more than 5%, no more than 2.5%, nomore than 1%, no more than 0.5%, no more than 0.1%, no more than 0.01%,or less. In one embodiment, the dry-state refers to 0% water content(i.e., completely dry).

In some embodiments where the silk-based implantable system and/ordevice is in a tubular form, e.g., a silk-based implantable electrode,the phrases “sufficient stiffness to penetrate a target tissue” and“stiff enough for insertion” can refer to a silk-based implantablesystem and/or device described herein having a Young's modulussufficient to yield a buckling force greater than a force typically usedto insert the silk-based implantable system and/or device into a targettissue (through a protective tissue layer). Thus, the implantable systemand/or device can remain substantially straight during insertion inorder to precisely reach a target region of the tissue. For example, thesilk-based implantable system and/or device described herein can have aYoung's modulus sufficient to yield a buckling force at least about 10%(including at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90% or higher) greater than a force used toinsert the silk-based implantable system and/or device into a targettissue (through a protective tissue layer). As used herein, the term“buckling force” refers to the maximum force required to cause a suddenfailure of a structural member (e.g., a silk-based implantable systemand/or device) subject to a compressive stress. In general, the bucklingforce of a structural member can be readily measured by a skilledartisan. For example, the buckling force of the silk-based implantablesystem and/or device can be measured by the buckling force testdescribed in the Examples. In some embodiments, the silk matrix asdescribed herein can increase a buckling force of the electricalcomponent or the implantable device by at least about 2-fold, at leastabout 5-fold, at least about 10-fold or more, as compared to the absenceof the silk matrix. In some embodiments, the silk matrix can increase abuckling force of the electrical component or the implantable device byat least about 1 order of magnitude (e.g., about 10-fold or more), about2 orders of magnitude (e.g., about 100-fold or more), about 3 orders ofmagnitude (e.g., about 1000-fold or more) or more, as compared to theabsence of the silk matrix.

In accordance with an aspect described herein, a silk-based implantablesystem comprises one or more electrical component (including two or moreelectrical components). As used herein, the term “electrical component”can include any component that is involved in an electric circuit, e.g.,for use as a sensor, a detector, a recording device, a current/voltagegenerator, and/or receiver. In particular embodiments, the electricalcomponent used herein are implantable and biocompatible in vivo.Exemplary electrical components can include, but are not limited to, anelectrode or electrode contact, a transistor, a resistor, a battery,conducting wires, a signal receiver, a signal transmitter, and anycombinations thereof.

In some embodiments, the electrical component can be at least part of apre-formed or a conventional implantable device that would generallydeform during the penetration in the absence of the silk matrix.Accordingly, in such embodiments, coating the preformed or conventionalimplantable device can provide the respective device with mechanicalstiffness sufficient to penetrate into a target tissue (e.g., withoutdeformation such as changing shape, buckling or bending). Uponpenetration, the silk coating can become softer or flexible, thusallowing the implantable device to be mechanically compliant with thetarget tissue.

A pre-formed or conventional implantable device can be an implantabledevice, e.g., an implantable electrical device, designed for any use inany part of the body in a subject, including, but not limited to,subcutaneous, intramuscular, intraperitoneal, cardiac, pulmonary, andneural tissue. In some embodiments, the pre-formed or conventionalimplantable device (e.g., an implantable electrical device) can be aneuroprosthetic device. Exemplary neuroprosthetic device can include,but are not limited to, a brain penetrating electrode, a shunt, a nerveguide, a cochlear implant, and a microelectrode array.

In some embodiments, the implantable electrical device can include aflexible electrode (e.g., an electrode that is not mechanically stiffenough to penetrate through a protective layer into a tissue). By way ofexample only, flexible electrodes can be fabricated by encapsulatinggold traces in parylene (See, e.g., Metallo C. et al., “Flexibleparylene-based microelectrode arrays for high resolution EMG recordingsin freely moving small animals,” Journal of Neuroscience Methods. (2011)Feb. 15; 195(2):176-84).

Design of a flexible electrode can vary with applications and/or desiredsizes. In some embodiments, the arrangement and/or shape of an electrodecan be adapted to decrease surface area, e.g., due to space and/shapeconstraint of a penetration site. The flexible electrode can then be atleast partly coated or encapsulated in silk. Once encapsulated, the silkproperties can be controlled for transition from stiff to flexible.

In some embodiments, the commercially-available flexible electrodes canalso be used in the implantable system described herein.

While the electrical component can be an integral part of a pre-formedor conventional implantable device, the electrical component can also bedirectly or indirectly patterned or deposited on the silk matrix to formthe silk-based implantable device. An exemplary patterning method isdescribed, e.g., in Kim, D. H., et al “Silicon electronics on silk as apath to resorbable implantable devices.” (2009) Applied Physics Letters95(13):133701; and Kim, D. H. et al. “Dissolvable films of silk fibroinfor ultrathin conformal biointegrated electronics.” (2010) NatureMaterials 9(6):511-517. These reports indicate that silicon transistorscan be fabricated on resorbable silk films for brain recordings on cats,with no inflammatory response in vivo.

The electrical component can be formed from any conventional materialscommonly used for electronics in an implantable device, e.g., withoutlimitations, silicon, gold, platinum, titanium, copper, alloys, and anycombinations thereof. In some embodiments, the electrical component doesnot include a silicon transistor. Alternatively, the electricalcomponent can include biodegradable electronics, e.g., made frombiodegradable or erodible organic semiconductors such as melanins andcarotenoids. In one embodiment, the electrical component can include asilk-based electrode as described herein.

In accordance with various aspects described herein, the silk matrix isdynamic with respect to its changes in the mechanical property before orduring and after penetration (or between a dry state and a hydratedstate). Before penetration and during at least part of the penetrationprocess, the silk matrix is stiff enough to enable the implantabledevice to penetrate into a target tissue (e.g., without deformation suchas buckling or bending). In some embodiments, the silk matrix canincrease a buckling force of the electrical component and/or theimplantable device by at least about 2-fold, at least about 3-fold, atleast about 4-fold, at least about 5-fold, at least about 10-fold, atleast about 25-fold, at least about 50-fold, at least about 100-fold, atleast about 250-fold, at least about 500-fold, at least about 1000-fold,at least about 1500-fold, at least about 2000-fold, at least about3000-fold, or higher, as compared to the absence of the silk matrix.

Upon penetration into the target tissue, the silk matrix in contact withthe electrical component can become compliant upon the penetration,e.g., by hydration of the silk matrix. Such mechanical compliance of thesilk matrix with surrounding tissue upon penetration can provideconformal contact between the electrical component and a surface of thetarget tissue. In some embodiments where the electrical component isassociated with a neuroprosthetic device (e.g., an electrode pad), atight interface can be formed between the neurons and the electrodepads. As used herein, the term “hydration” or “hydrated state” refers toat least a portion of the silk matrix having a water content of at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, or more.

As used herein, the term “conformal contact” generally refers to acontact established between two surfaces. In one embodiment, conformalcontact involves a macroscopic adaptation of one or more contactsurfaces of an implantable system (including the electrical componentand the silk matrix) or device to the overall shape of the surroundingtissue. In another embodiment, conformal contact involves a microscopicadaptation of one or more contact surfaces of an implantable system(including the electrical component and the silk matrix) or device tothe surrounding tissue leading to an intimate contact without anydetectable gap or a gap that would affect electrical connectivitybetween the device and the tissue (e.g., less than 50 μm, less than 40μm, less than 30 μm, less than 20 μm, less than 10 μm, less than 5 μm,less than 1 μm, less than 0.5 μm, less than 0.1 μm, less than 0.01 μm orless).

In some embodiments, at least one side of the electrical component canbe coated with the silk matrix. Thus, at least part of the electoralcomponent can still be exposed to surrounding tissue, e.g., fordetecting a signal. In some embodiments, the electrical component can bepartially or completely coated or encapsulated in a silk matrix. Withoutwishing to be bound, the electrical component can also be encapsulatedin the silk matrix. For example, in the case of an electrodeencapsulated in a silk matrix, the silk around the electrode tip caneither degrade immediately, or contain holes allowing close contact withthe electrodes. Alternatively, the silk matrix over the electrode can bemodified, doped, or functionalized with a conducting polymer (Abidian M.R. et al., “Multifunctional Nanobiomaterials for Neural Interfaces”Advanced Functional Materials (2009) 19: 573-585), thus making the silkmatrix become conductive and thus extending the electrode through thesilk.

In some embodiments, the silk matrix can reduce scar formation (e.g.,gliosis) around the electrical component or the implantable device by atleast about 10%, including at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95% orhigher, as compared to the absence of the silk matrix.

Silk matrix can stabilize an active agent including a therapeutic agentfor an extended release in vivo. Accordingly, in some embodiments, thesilk matrix can comprise an active agent including, but not limited to,a therapeutic agent. The active agent (including a therapeutic agent)can include, e.g., but are not limited to, an agent that promotes tissuegrowth, controls inflammation, and/or reduces scar formation around theimplanted system or device. In one embodiment, the active agent caninclude a gliosis-modulating agent. In some embodiments, the silk matrixcan comprise two or more (e.g., 2, 3, 4, 5, 6 or more) active agentsincluding therapeutic agents.

Depending on applications of the implantable device, and/or mechanicalproperty of the silk matrix and/or implantation sites, the silk matrixcan have a thickness of about 1 μm to about 1000 μm, about 5 μm to about750 μm, about 10 μm to about 500 μm, about 25 μm to about 250 μm, orabout 50 μm to about 200 μm. In some embodiments, the silk matrix canhave a thickness more than 1 mm, including, e.g., more than 1.5 mm, morethan 2 mm, or more than 3 mm, provided that the thickness can stillprovide mechanical compliance with the surrounding tissue uponpenetration. The total thickness of the silk matrix can be resulted froma single layer or a plurality of silk layers, each of which can have thesame or different thickness and optionally the same or different activeagent. In some embodiments, the silk matrix can have at least about 1layer, at least about 2 layers, at least about 3 layers, at least about4 layers, at least about 5 layers, at least about 10 layers, at leastabout 15 layers, at least about 20 layers, at least about 25 layers, atleast about 50 layers, at least about 100 layers or more. In oneembodiment, the silk matrix can have at least about 3 layers to about 6layers. Each layer can have a thickness of about 1 μm to about 100 μm,about 5 μm to about 75 μm or about 10 μm to about 30 μm. In someembodiments of any aspects described herein, the silk matrix in contactwith an electrical component can have a thickness greater than 5 μm,greater than 10 μm, greater than 20 μm, greater than 30 μm, greater than40 μm, greater than 50 μm or more. In some embodiments, the silk matrixin contact with an electrical component can have a thickness rangingfrom about 20 μm to about 1000 μm, about 30 μm to about 900 μm, about 40μm to about 800 μm, about 50 μm to about 700 μm, about 50 μm to about600 μm, or about 100 μm to about 500 μm.

The silk matrix coating on, depositing on, and/or encapsulating theelectrical component and/or the implantable device can be performed byany methods known in the art, e.g., by dipping, casting and/orlayer-by-layer deposition. In one embodiment, an electrical componentand/or at least part of the implantable device can be dipped into a silkfibroin solution at a concentration of about 3% w/v to about 30% w/v, orabout 5% w/v to about 25% w/v, or about 10% w/v to about 20% w/v. Thecoating is then allowed to dry. For example, as shown in 9A, the dipcoating approaches can involve briefly immersing a pre-formedimplantable device in a silk solution, followed by air-drying. Thesolutions utilized for dip coating can include (i) aqueous silksolution, (ii) high silk concentration HFIP solution, and (iii)heat-liquefied silk electro-gel (e-gel) solution, wherein the e-gel canhave a transition between a gel and a solution upon application of avoltage. See the PCT Application No. PCT/US2009/058534 filed Sep. 28,2008 for information about e-gel, the content of which is incorporatedherein by reference.

Alternatively, casting methods can involve submerging a pre-formedimplantable device in a volume of aqueous or gel based silk solution andallowing the silk to air dry to form films (see FIGS. 6B-6C). The coatedimplantable device can then be manually cut out of the encapsulatingfilm using a razor blade or scissors. For more precise release of thecoated device from the silk film, a computer controlled laser cutter canbe employed.

In particular embodiments, the casting method can involve a two-steplayering process. First, a volume of heat-liquefied silk e-gel can bepoured into a plate and allowed to re-gel by cooling (3-5 minutes). Theimplantable device and/or electrical component to be encapsulated canthen be placed on top of the first e-gel layer and covered by anothervolume of heat-liquefied e-gel. The layered gel can then be left to airdry. This process can ensure even layers of silk on both sides of theelectrical component and/or the implantable device, preventingdelamination of the silk from the substrate upon drying. The coatingformed uniform layers on the electrical component and/or the implantabledevice, the thickness of which can be individually controlled byadjusting the volume and/or concentration of the silk solution appliedin each layer (see FIGS. 7A-7B and 8). Furthermore, the viscosity of theliquefied e-gel solution can allow for selective and localizedapplication of the top layer. This can enable portions of theimplantable device to be left uncoated as desired, e.g., electricalleads for contact with surrounding cell(s) and/or tissue uponpenetration. For instance, as shown in FIG. 9, only one side of thecontact pads on an electrode was coated with silk. In some embodiments,an active agent can be incorporated into a silk solution, which can beentrapped upon drying and released after implantation.

Applications of the silk-based implantable systems are also providedherein. For example, a method of inserting a flexible or softimplantable device (e.g., an implantable device that generally bucklesand/or bends during insertion) into a target tissue comprises providinga silk-based implantable system described herein, wherein the silk-basedimplantable system comprises the flexible or soft implantable device atleast partially coated with a silk matrix in its dry-state. Thethickness of the silk matrix coating on the flexible or soft implantabledevice can vary with the penetrate site and/or mechanical property ofthe target tissue to be penetrated. In some embodiments, the thicknessof the silk matrix coating on the flexible or soft implantable devicecan be adjusted such that the silk-coated implantable device is stiffenough to penetrate into the target tissue, but become compliant withthe target tissue within a period of time (e.g., within minutes orhours) after penetration.

In some embodiments, the silk-based implantable systems can be used toreduce scar formation (e.g., gliosis) around the implantable device(e.g., a neuroprosthetic device) implanted in a tissue (e.g., a braintissue), for example, by at least about 5%, at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or more, as compared to an implantabledevice without a silk coating. Accordingly, a method for reducing scarformation (e.g., gliosis) around an implantable device (e.g., aneuroprosthetic device) implanted in a tissue (e.g., a brain tissue) isalso provided herein, e.g., by providing or obtaining a silk-basedimplantable system described herein or silk-based implantable devicedescribed below for implantation in a target tissue. In some embodimentswhere the pre-formed or conventional implantable device may not requirea silk coating for additional strength during penetration, the silkmatrix coating can be formed in situ, for example, the silk coating canbe formed at the target site upon penetration. In such embodiments, thepre-formed or conventional implantable device can be adapted to includea silk reservoir which can supply a silk solution for forming in situcoating.

In some embodiments, the silk-based implantable systems can be used toimprove or extend long-term functionality of an implantable device(e.g., a neuroprosthetic device) implanted in a tissue (e.g., a braintissue) by at least about 3 days, at least about 1 week, at least about2 weeks, at least about 3 weeks, at least about 1 month, at least about2 months, at least about 3 months, at least about 4 months, at leastabout 5 months, at least about 6 months or more, as compared to theimplantable device without a silk coating. For example, the silk-basedimplantable system can reduce biofouling around an implanted device,e.g., by at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, ormore, as compared to an implanted device without a silk coating. Withoutwishing to be bound by theory, biofouling can, at least partly,contribute to loss of function (e.g., recording of an electric signal ata biologically-relevant level, and/or providing an electricalstimulation) of an implantable device at the biological abioticinterface. Accordingly, reducing biofouling, e.g., by having a silkmatrix coating on the implantable device can extend the life-time and/oroperation time of the implantable device upon penetration in a tissue byat least about 1 week, about 2 weeks, about 3 weeks, about 1 month orlonger. In some embodiments, the silk matrix coating can be pre-formedon the implantable device and/or be formed in situ as described herein.

Silk-Based Implantable Devices and Methods of Making the Same

Electronics that are capable of intimate, non-invasive integration withthe soft, curvilinear surfaces of biological tissues offer opportunitiesfor monitoring, diagnosing and treating disease or injury and forestablishing brain/machine interconnects. Previous report shows abio-interfaced system that relies on ultrathin film electronicssupported by a bioresorbable substrate of silk. Placing such device on atissue and then allowing the silk to dissolve and resorb can initiate aspontaneous, conformal wrapping process driven by capillary forces atthe biotic/abiotic interface. Specialized mesh designs and ultrathinforms for the electronics can ensure minimal stresses on the tissue andhighly conformal coverage, even for complex curvilinear surfaces. See,e.g., Kim et al. “Dissolvable films of silk fibroin for ultrathinconformal bio-integrated electronics” Nature Materials (2010) 9:511-517. However, the report shows direct placement of suchsilk-supported ultrathin film electronics on a surface of a braintissue, rather than inserting such device into a brain tissue. Such silkfilm-supported planar arrays generally use low silk concentrations,e.g., less than 5% (w/v) for thin sheets, that will conform toconvoluted surfaces. Thus, unlike the silk-based implantable systemsdescribed herein, the silk film-supported planar arrays do not have themechanical strength necessary for insertion into a tissue. Further,unlike some embodiments of the silk-based implantable systems describedherein, theses thin films are designed to dissolve immediately, and thusthey are not suitable for encapsulation of an active agent for anextended release.

One of the challenges with implantable devices is the effect ofbio-fouling. For example, electrodes used for deep brain stimulation cangenerally function in the brain after implantation for a limited of timebecause of the bio-fouling. Another aspect described herein relates to asilk-based implantable device comprising a silk body with at least oneelectrically-conducting component. In some embodiments, the silk-basedimplantable device can be constructed to be capable of renewably forminga silk coating on a surface of the implantable device. Such silk-basedimplantable device can minimize biofouling, thus increasing thereliability and lifetime of the implantable device in vivo.

For example, in some embodiments, the silk body can include a silkreservoir of any shape with at least one electrically-conductingcomponent formed on a least a portion of a surface of the silk reservoir(e.g., the surface that is in contact with a fluid upon implantation).In some embodiments where the silk-based implantable device are adaptedfor use as an electrode, the silk-based implantable device can comprisea silk body with more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 25,50, 75, 100, 250, 500, 750, 1000, 1500 or more) electrically conductingcomponents. The term “electrically conducting component” as used hereinrefers to any component that is involved in an electric circuit toconduct electricity, e.g., but not limited to, electrodes, transistors,capacitors, battery leads, and/or electrical connectors such as wires ormicrowires.

In some embodiments, the electrically conducting component includes anelectrode. As used herein, the term “electrode” means an electronicincluding an electric conductor through which a voltage potential can bemeasured. An electrode can also be a collector and/or emitter of anelectric current.

In some embodiments, the term “electrode” as used herein refers to theelectrode or electric contact or contacts only. In some embodiments, theterm “electrode” can refers to the electrode or electric contact orcontacts and one or more surrounding structures, e.g., a supportsubstrate upon which the contacts are placed, the conductor wires andany other assemblies within or on the support substrate.

The silk body can be in any shape and/or form, depending on theapplication of an application device, and/or implantation site in abody. For example, the silk body can have a curved or a planar surface.In one embodiment, the silk body can be in a form of a tube, e.g., foruse as an implantable electrode. Accordingly, in some embodiments, asilk-based implantable device comprising a silk-based self-cleaning orliving-like electrode is also provided herein.

The silk body can have a lumen extending therethrough or one or morecompartments independently distributed within a silk body for use as asilk reservoir. The lumen can have the same cross-section as that of thesilk body or a cross-section that is different than that of the silkbody. For example, the cross-section of the lumen can be round,substantially round, oval, substantially oval, elliptical, substantiallyelliptical, triangular, substantially triangular, square, substantiallysquare, hexagonal, substantially hexagonal, or the like.

In some embodiments, the lumen has a diameter. The diameter can be, forexample, approximately the same as the diameter of the rotating mandrelused in the preparation of the silk body. It is understood that thediameter can vary along the length of the lumen. Without limitations,the diameter can be from about 100 nm to about 10 mm. In someembodiments, the diameter can be from about 1 mm to about 5 mm, fromabout 1 mm to about 3 mm, from about 3 mm to about 5 mm, from about 2 mmto about 4 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, or about5 mm. In some embodiments, the diameter can be greater than about 5 mm.In some embodiments, the diameter can be less than about 1 mm. In otherembodiments, the diameter of less than about 20 mm, for example, lessthan about 10 mm, or less than about 5 mm.

In some embodiments, the silk body can be a silk tube with at least one(including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or more)electrically-conducting component formed on at least a portion of alateral surface of the silk tube. For example, theelectrically-conducting components can be arranged orderly or randomlyon the lateral surface of the silk tube. In some embodiments, theelectrically-conducting components are arranged on pre-defined locationson the lateral surface of the silk tube.

FIG. 12A shows one embodiment of a silk-based self-cleaning orliving-like electrode. As shown in FIG. 12A, the silk-based electrodeincludes a silk tube with one or more electrically-conducting componentsformed on at least a portion of a lateral surface of the silk tube. Inthis embodiment, the silk tube is filled with a silk solution. The silksolution in such embodiments can be of any concentration that can form acoating sufficient to reduce biofouling or reduce gliosis as discussedherein. In some embodiment, the silk solution filled inside the silktube can be at a concentration of about 10% (w/v) to about 30% (w/v), orabout 15% (w/v) to about 25% (w/v). In some embodiments, the silksolution filled inside the silk tube can have a concentration of about15% (w/v) or greater. Without wishing to be bound, in some embodiments,the silk concentration inside the silk tube can be lower than 15% (w/v)or lower than 10% (w/v), e.g., when the silk coating formed on theoutside surface of the electrode is desirable to be thin and degradeover a short period of time. The silk tube can be very tough (e.g., withsufficient mechanical stiffness to penetrate a target tissue), but alsowith the ability to soften when placed in a wet environment.

Generally, the silk body (e.g., a silk tube) can have any lengthdesired. For example, length of the silk body (e.g., a silk tube) can befrom about 1 mm to about 10 cm. In some embodiments, length of the silkbody (e.g., a silk tube) can be from about 1 mm to about 5 cm.

Without wishing to be bound by a theory, wall thickness of the silk tubeand/or silk reservoir can affect the mechanical property of the implantand/or release of the silk solution encapsulated therein. Accordingly,the silk tube and/or silk reservoir can be selected to have a wallthickness that provides a desired rate of release. For example, wallthickness can range from about 50 μm to about 5 mm. In some embodiments,the wall thickness can be from about 50 μm to about 500 μm, from about50 μm to about 1,000 μm, from about 200 μm to about 300 μm, from about600 μm to about 800 μm, from about 200 μm to about 800 μm, from about300 μm to about 700 μm, from about 400 μm to about 600 μm, or about 500μm. In some embodiments, the wall thickness can be greater than about1,000 μm. In some embodiments, the wall thickness can be less than about100 μm. In some embodiments, the wall thickness can be about 0.25 mm,about 0.5 mm, about 0.75 mm, about 0.9 mm, about 1.0 mm, or about 1.7mm.

As shown in FIG. 12A, in some embodiments, molding and shaping canprovide the silk tube with a sharp tip and the material is stiff enoughto be placed into a human brain without bending or fracturing.Circumferential electrodes can be formed on the outer tube surface bydepositing a thin layer (tens of micrometers) of noble metal (e.g.,gold) at predefined positions. Electrical leads can be connected throughthe inner diameter of the silk reservoir. Solubilized silk can besupplied through the inner tube diameter and discharged from theperforations in the electrodes. Electrogelation of the silk solution onthe electrode surface through the application of a low DC voltage canprovide a thin gel barrier to prevent bio-fouling, without degradingelectrode function. Incorporation of the silk electrogelation(sol-->gel) process is an exemplary feature of the silk-basedimplantable device described herein, because a polarity change involtage application can causes the silk gel coating to dissipate(gel-->sol). In this way, the electrode surface can be refreshed withthe removal of bio-foul buildup and replacement of a fresh silk gelcoating to restore good electrical connections. Electrogelation of silkis described in PCT application: PCT/US2009/058534, the content of whichis incorporated herein by reference. While FIG. 12A shows an entirelysilk based implant with electrical contacts, methods of regenerating asilk coating can be applied to any implant of any material withelectrical contacts. For example, an implant can be adapted to include areservoir for silk solution, which can be in transient connection withan electric lead formed a surface of the implant to be coated.

The silk body can be formed from any concentration of silk, depending onthe desired mechanical property of the implantable device, e.g., rangingfrom 5% w/v to about 40% w/v. Generally, the higher the concentration ofsilk is used, the stiffer the dry-state silk body will be. In someembodiments, the silk body can be formed from a high-concentration silksolution, e.g., ranging from about 10% w/v to about 40% w/v, or from 15%w/v to about 30% w/v. In some embodiments, the silk body can be formedfrom a silk solution with a concentration higher than about 20% w/v.

Generally, silk tubes can be made using any method known in the art. Forexample, tubes can be made using molding, dipping, spinning, and anycombinations thereof. In one embodiment, the silk tube can be formedfrom a silk solution using a cylindrical mold (e.g., slightly oversizedthan the final desired outcome to accommodate shrinkage) and removablewire core. The solid material can then be removed from the mold andmaintained at room temperature for 3 days (depending on the humidity)until the material is nearly dry. The center core is then removed andthe silk tube is fully dried.

Alternatively, the silk tube can be produced from a silk solution byspinning a silk solution around a rotating and optionally axiallyreciprocating support structure (e.g., a mandrel). The term “spinning”as used herein can encompass any methods for creating a fiber,including, but not limited to, wet-spinning, dry-spinning,melt-spinning, e.g., extrusion spinning and direct spinning, gelspinning, electrospinning.

In some embodiments, the silk tube can be produced from a silk solutionby gel spinning. Gel spinning involves winding a silk solution around areciprocating and rotating mandrel. As shown in FIG. 16, final gel-spunsilk tube porosity, winding patterns, structure and mechanicalproperties can be controlled via axial and/or rotational speeds of themandrel and/or different post-spinning processes such as alcohol (e.g.,methanol, ethanol, etc) treatment, air-drying or lyophilization. Gelspinning is described in Lovett et al. (Biomaterials, 29(35):4650-4657(2008)) and in PCT application no. PCT/US2009/039870, entitled “Systemand Method for Making Biomaterial Structures” filed Apr. 8, 2009, thecontent of all of which is incorporated herein by reference. Withoutwishing to be bound by a theory, the inner and outer diameter of thesilk tube can be controlled readily using gel-spinning.

In some embodiments, the silk tube can be produced from a silk solutionby electrogelation. For example, the silk solution can become gel-likearound a tubular support structure upon application of a voltage to thesupport structure. Electrogelation of a silk solution is described inPCT application no. PCT/US2009/058534 filed Sep. 29, 2008, the contentof which is incorporated herein by reference.

Any other art-recognized methods for making a tubular structure can alsobe used to the silk tubular body. For example, the method described inU.S. application Ser. No. 12/672,521, entitled “Tubular SilkCompositions and Methods of Use Thereof” filed Aug. 11, 2008 can also beused for making a silk tubular body.

In some embodiments, the tubular structure can be produced bymolding/freezing method. For example, a silk solution of about 25% w/vfibroin can be injected into a tubing as a mold and both ends can besealed, e.g., using the high heat of a soldering iron. Thesilk-containing tubing can then be stored for a pre-determined period oftime at a sub-zero temperature, e.g., between about −3° C. and about −9°C. In some embodiments, the silk-containing tubing can be stored at −5°C.

The resulting morphology of the silk matrix can depend on, e.g., thecooling temperature and/or duration of cooling. In some embodiments, thesilk-containing tubing can be stored at a sub-zero temperature for atleast about 3 days, at least about 1 week, at least about 2 weeks, atleast about 3 weeks, at least about 4 weeks or longer. In oneembodiment, the silk-containing tubing is stored at about 5° C. for aperiod of at least about 1 week.

Upon removal from the freezer, the molded material can released from thetube by any methods known in the art, e.g., by flushing the innerdiameter with a fluid, e.g., water, or cut the mold open.

Such molding/freezing processing method can allow fabrication of tubularstructure with any diameters, e.g., by using an appropriate tubingselection. Further, the molding/freezing processing method can be usedto produce a silk body of different shape other than a tubularstructure, e.g., an irregular shape, depending on the shape and/or formof the mold. An exemplary molding/freezing processing of a silk solutionto form different structures is described, e.g., in Prov. App. No.61/477,486, filed Apr. 20, 2011, the content of which is incorporatedherein by reference.

In some embodiments, the silk body can comprise a first silk layer and asecond silk layer, wherein at least one of the first and the second silklayer can comprise at least one electrically-conducting component formedon at least a portion of a surface of the silk layer. In someembodiments, silk particles or powder can be included between the firstand the second silk layers. In some embodiments, the silk body can havemore than 2 layers, e.g., 3, layers, 4 layers, 5 layers, 6, layers, 7layers, 8 layers, 9 layers, 10 layers, 15 layers, 20 layers or more. Thenumber of silk layers can affect the thickness of the silk body and/ormechanical property of the silk body. In general, increasing the numberof silk layers can increase the thickness of the silk body and thusstrengthening the mechanical property of the silk body, when it is in adry-state. Further, increasing the number of silk layers can increasethe period of time required to make the silk body become flexible and/orsofter.

The silk layers of the silk body can be formed by layer-by-layerdeposition method. For example, a second silk layer is independentlyformed or deposited on the first silk layer. Alternatively, the silklayers of the silk body can be formed by folding, rolling or wrapping asilk sheet one or a plurality of times to reach a desired number oflayers.

FIG. 12B shows an example of a silk-based self-cleaning or living-likeelectrode formed by wrapping or rolling a silk sheet. Electrodes can beformed on the silk paper before forming a tube by depositing a thinlayer of noble metal (e.g., gold) at specific positions. Metaldeposition can also be employed to make electrical connections to theelectrodes on silk paper. While the electrode design does not involvethe introduction of a silk solution from outside the body, silk solutioncan be produced in situ. To achieve this, lyophilized silk particles,powder, gel-like particles, silk film, or gel-like silk film can bewrapped between the layers of silk sheet. Upon introduction of the silkelectrode into a tissue (e.g., brain tissue), the presence of aninterstitial fluid or body fluid surrounding the target tissue (e.g.,cerebrospinal fluid) can cause the outermost lyophilized silk tosolubilize. Similar to the silk-based electrode shown in FIG. 12A, thissolubilized silk can then be discharged through perforations in theelectrodes and form a thin gel coating through the electrogelationprocess. In some embodiments, the silk-based electrode as shown in FIG.12B is designed to be sacrificial. As the underlying silk sheet degradesover time, starting with the outer layer, fresh electrode surfacesbecome exposed. In this way, the silk electrode can have an extendeduseful lifetime.

In any embodiments, the discharge of a silk solution from a silkreservoir onto an electrically-conducing component of the silk body canbe controlled by any methods known in the art, e.g., by passivediffusion, an implantable pump and/or an external pump. Passivediffusion of silk solution can be partly controlled by beta-sheetcontent, and/or concentration of the silk solution.

In various embodiments of the silk-based implantable devices describedherein, to allow the silk solution to form a coating on a surface of theelectrically-conducting component, in some embodiments, theelectrically-conducting component can include one or more through holessuch that the silk solution present in the silk reservoir (e.g., silktube) between the silk layers can be discharged onto the surface of theelectrically-conducting component to be coated. The solution can bedischarged onto the surface of the electrically-conducting component tobe coated by any methods known in the art, including, but not limitedto, diffusion, an implantable pump, and/or an external pump. The silksolution discharged onto the surface of the electrically-conductingcomponent can form a gel-like coating upon application of a firstvoltage through the electrically-conducting component. In someembodiments, the gel-like coating can be removed, e.g., by transformingthe gel-like coating to a solution upon application of a second voltagewith a polarity opposite to the first voltage.

The electrical conducting component can include any material that iscommonly used as electronics for implantable devices, and/or anelectrically-conductive material. In some embodiments, the electricalconducting component can include a metal such as a transition metal(e.g., silicon, copper), a noble metal (e.g., gold, titanium, platinum),or a combination thereof.

In some embodiments, the electrical conducting component can include abiodegradable component that can conduct electricity such asbiodegradable organic semiconductors (e.g., melanins and/orcarotenoids).

In some embodiments, the electrical conducting component can includesilk modified to conduct electricity. For example, the silk can befunctionalized by modifying a tyrosine of the silk protein to a sulfategroup followed by polymerization of the modified tyrosine with aconducting polymer. Alternatively, the silk can be doped with aconductive material including, but not limited to, gold nanoparticles,carbon nanotubes, graphene, and a conducting polymer. Non-limitingexamples of a conducting polymer can include polyethylenedioxythiophene(PEDOT), polypyrrole-based conductive polymer, copolymers of thiophenesand polypyrroles, copolymers of poly-lactide and polyaniline, or anycombinations thereof. Exemplary methods for modifying silk to conductelectricity are further described later.

In some embodiments, the silk-based implantable device can be adaptedfor use as an implantable brain penetrating electrode. For example, thesilk body can be in a form of a silk tube with a diameter of less than 2mm. In some embodiments, the silk-based electrode can have a tensilestrength of at least about 2 MPa when the silk body is in a dry state.In some embodiments, the silk-based electrode can have a shear modulusof less than about 200 kPa upon contact of the silk body with a fluid(e.g., interstitial fluid and/or body fluid such as cerebrospinalfluid).

In some embodiments, the silk body can comprise one or more activeagents including a therapeutic agent. In some embodiments, the silk bodycan comprise two or more active agents including one or more, or two ormore therapeutic agents.

Methods for regenerating a silk coating on a surface of a device arealso provided herein. In some embodiments, the method comprisesproviding the silk-based device described herein, wherein the silksolution discharged onto the surface of the electrically-conductingcomponent can form a gel coating upon application of a first voltagethrough the electrically-conducting component, and can optionally turnto a solution upon application of a second voltage with a polarityopposite to the first voltage.

The capability of renewing or regenerating the silk coating on a surfaceof a device can reduce biofouling, e.g., by at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 95%, or more, as compared to an implanteddevice without a silk coating. Accordingly, a method of reducingbiofouling of a device is provided herein. The method comprisesproviding the silk-based device described herein, wherein the silksolution discharged onto the surface of the electrically-conductingcomponent forms a gel coating upon application of a first voltagethrough the electrically-conducting component, and can optionally turnto a solution upon application of a second voltage with a polarityopposite to the first voltage.

In some embodiments of any methods described herein, the first voltageand/or the second voltage can be applied to the electrically-conductingcomponent at any potential, provided that the voltage potential is highenough for silk gelation, but not detrimental to the tissue in contactwith the electrically-conducting component. In some embodiments, thefirst voltage and the second voltage can be at least about 1.2V, atleast about 1.5V, at least about 2V, at least about 3V, at least about5V, at least about 10V, at least about 25 V, at least about 30V, atleast about 50V, at least about 75 V or higher. In other embodiments,the first voltage and the second voltage can be about 1.2V to about100V, about 2 V to about 75V, or about 5 V to about 50 V.

In some embodiments of any method described herein, the silk solutiondischarged onto the surface of the electrically-conducting component toform a gel coating can occur once a week, once a month, once every twomonths, or once every three months, or less frequently.

In some embodiments, the methods described herein can further comprisingsolubilizing the previous gel coating formed on the surface of theelectrically-conducting component, e.g., prior to forming another freshlayer of the gel coating, by applying a second voltage with a polarityopposite to the first voltage. Accordingly, in some embodiments, thecapability of the silk-based implantable devices to renew or regeneratea silk coating on a surface of the device can extend the life-timeand/or operation time of the implantable device upon penetration in atissue by at least about 1 week, about 2 weeks, about 3 weeks, about 1month or longer.

In some embodiments, the silk-based implantable device can be placed invivo or in situ. Thus, any embodiments of the methods described hereincan be carried out in vivo or in situ. Without wishing to be bound, notonly can different aspects of dynamic silk coating described herein beapplicable for in vivo use (e.g., in a subject in situ such as amammalian subject, e.g., human), but they can also be used in anon-living object in situ, e.g., in a machine.

Silk-Based Electrode

Some embodiments of the silk-based implantable device can be adapted foruse as silk-based electrodes in any part of a body in a subject. In oneembodiment, the silk-based implantable device described herein can beadapted for use as a silk-based brain penetrating electrode.

For example, the silk-based electrode can have a dimension large enoughto yield a mechanical stiffness sufficient to penetrate into a targettissue, while small or thin enough to avoid any significant tissuedamage. The target tissue can be anywhere in the body of a subject,e.g., where an electrical stimulation and/or signal recording is inneed. Exemplary target tissue can include, but are not limited to,neural tissue (e.g., brain dura), heart tissue, cochlea, cochlearnucleus complex in the lower brain stem, inferior colliculus, cornea,aqueous humor, vitreous humor, or spinal cord. In some embodiments, thesilk-based electrode can have a mechanical stiffness sufficient topenetrate into a brain tissue such as brain dura.

In some embodiments, the silk-based electrode can have a diameter ofless than 2 mm, less than 1 mm, less than 0.5 mm, less than 0.25 mm,less than 0.1 mm or smaller. In some embodiments, the silk-basedelectrode can have a diameter of less than 500 μm, less than 250 μm,less than 100 Gm, less than 50 Gm, less than 25 μm, less than 10 Gm,less than 5 Gm, less than 2.5 Gm, less than 1 Gm, less than 0.5 μm, orlower. Depending on the mechanical property of the target tissue, thesilk-based electrode can have a diameter of more than 2 mm or smallerthan 0.5 μm.

Human dura mater has been reported to have tensile strength ranging from3-12 MPa, with a Young's modulus ranging from 20-190 MPa, depending onfiber orientation (Zerris et al., 2007). A previous report shows a peakinsertion force of 140 mN for a silicon probe with thickness 100 μm andwidth 120 μm inserted through the dura of a monkey (Hoffmann et al.,2007). Accordingly, in some embodiments, in order to be sufficientlystiff to penetrate into the target tissue, the silk-based electrode canhave a Young's modulus of more than 1 MPa, 2 MPa, 3, MPa, 4 MPa, 5 MPa,6 MPa, 7 MPa, 8 MPa, 9 MPa, 10 MPa, 15 MPa, 20 MPa, 30 MPa, 40 MPa, 50MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, 100 MPa, or higher, when it is in adry-state. In some embodiments, the silk-based electrode can have aYoung's modulus of more than 100 MPa, 250 MPa, 250 MPa, 500 MPa, 1000MPa, 2500 MPa, 5000 MPa, 7500 MPa, 10,000 MPa, or higher, when it is ina dry-state. As used herein, the term “dry-state” refers to a silkmatrix being hydrated (e.g., water content) for no more than 30%, nomore than 20%, no more than 10%, no more than 5%, no more than 2.5%, nomore than 1%, no more than 0.5%, no more than 0.1%, no more than 0.01%.In one embodiment, the dry-state refers to 0% water content (i.e.,completely dry).

In accordance with different aspects provided herein, the silk-basedelectrode becomes compliant upon the penetration into the target tissue.As used herein, the term “compliant” refers to a silk-based implantablesystem or device becoming mechanically compliant to a target tissue uponpenetration into a target tissue. For example, the silk-basedimplantable system or device is able to reduce strain arisen from themismatch between the elastic modulus of brain tissue and the silk-basedimplantable system or device in its dry-state, e.g., by becoming softeror flexible when in contact with a fluid upon the penetration. By way ofexample only, the shear modulus of brain tissue is ˜10 kPa, with whitematter stiffer (˜12 kPa) than gray matter (˜8 kPa) (McCracken et al.,2005). A 0.6% agar gel is commonly used as a phantom to approximate themechanical stiffness of the brain.

Accordingly, in some embodiments, the silk-based electrode, upon thepenetration in a target tissue (and/or contact with a fluid) for acertain period of time, can have an elastic modulus reduced by at leastabout 2-fold, at least about 3-fold, at least about 4-fold, at leastabout 5-fold, at least about 10-fold, at least about 25-fold, at leastabout 50-fold, at least about 100-fold, at least about 250-fold, atleast about 500-fold, at least about 750-fold, at least about 1000-fold,as compared to when it is in the dry-state, e.g., before penetrationinto the target tissue. In some embodiments, the silk-based electrode,upon the penetration in a target tissue (and/or contact with a fluid)for a certain period of time, can have an elastic modulus or shearmodulus of less than 500 kPa, less than 250 kPa, less than 200 kPa, lessthan 150 kPa, less than 100 kPa, less than 75 kPa, less than 50 kPa,less than 25 kPa, less than 20 kPa, less than 10 kPa, less than 5 kPa orlower. In one embodiment, the silk-based electrode can have a shearmodulus of less than about 200 kPa upon contact of the silk body with afluid (including interstitial fluid and/or body fluid such ascerebrospinal fluid).

Without wishing to be bound by theory, the silk-based implantable systemor device (e.g., silk-based electrode) becomes softer or flexible whenit is hydrated by being in contact with a fluid (e.g., interstitialfluid or body fluid such as cerebrospinal fluid) upon penetration into atarget tissue for a certain period of time. Depending on the dimension,beta-sheet content of silk, silk concentration, structure/surface area(e.g., porous vs. non-porous), additives (e.g., glycerol) if any, and/ordry-state mechanical property of the silk-based implantable system ordevice, the silk-based implantable system or device can become softer orflexible upon penetration into a target tissue after a period of timeranging from few minutes to hours to days. In some embodiments, thesilk-based implantable system or device can become softer or flexibleupon penetration (e.g., become compliant with the target tissue to bepenetrated) after at least about 1 minute, at least about 2 minutes, atleast about 3 minutes, at least about 5 minutes, at least about 10minutes, at least about 15 minutes, at least about 30 minutes or longer.In some embodiments, the silk-based implantable system or device canbecome softer or flexible upon penetration (e.g., become compliant withthe target tissue to be penetrated) after at least about 1 hour, atleast about 2 hours, at least about 3 hours, at least about 4 hours, atleast about 5 hours, at least about 6 hours, at least about 12 hours, atleast about 16 hours, at least about 24 hours, at least about 2 days, atleast about 3 days, at least about 4 days, at least about 5 days, atleast about 6 days, at least about 7 days or more. In some embodiments,the silk-based implantable system or device can become softer orflexible upon penetration into a target tissue after a period of about16 hours to about 30 hours. In some embodiments, the hydration orre-hydration rate can be further controlled by addition of a morehydrophobic coating layer (e.g., higher beta sheet content), or polymeradditions that can be used to further control the rehydration and theresulting mechanical properties.

Silk is 100% degradable via enzymatic processes, with programmablelifetimes from hours to years pending the mode of processing (See, e.g.,Wang, X. et al. “Controlled release from multilayer silk biomaterialcoatings to modulate vascular cell responses.” Biomaterials (2008)29(7):894-903; Kim, D. H., et al. “Dissolvable films of silk fibroin forultrathin conformal biointegrated electronics. Nature Materials” (2010)9(6):511-517). The degradation rate of the silk matrix within thesilk-based implantable system or device as described herein can becontrolled by the beta-sheet crystalline content of silk, silkconcentration and/or porosity of the silk matrix. Generally, increasingthe beta-sheet crystalline content of silk and/or silk concentration,and/or decreasing porosity of the silk matrix can decrease thedegradation rate of the silk matrix within the silk-based implantablesystem or device. Methods to increase the beta-sheet crystalline contentof silk is known in the art, e.g., by alcohol immersion, waterannealing, heat annealing, electrogelation, pH, shear stress, and anycombinations thereof. For example, water vapor annealing at variabletemperatures can be used to directly control crystalline content (betasheet) which relates directly to degradation lifetime.

In the case of degradation and/or dissolution, Protease XIV can beutilized to assess degradation kinetics under physiological conditionsat 37° C. Controls can be run under the same conditions but withoutenzyme. Gravimetric weight changes can be used to track changes withtime. This process can be regulated so that degradation is almostimmediate (e.g., dissolution) due to a low beta sheet content, or long(weeks, months or years), if beta sheet content is higher. If needed,inhibitors of proteases can be included in devices to further controlthe degradation process.

In some embodiments, the silk matrix within the silk-based implantablesystem or device (e.g., silk-based electrode) can partially orcompletely (e.g., at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, up to and including 100% of the silk matrix) degrade within atleast about 1 day, at least about 2 days, at least about 3 days, atleast about 4 days, at least about 5 days, at least about 6 days, atleast about 7 days or more. In some embodiments, the silk matrix withinthe silk-based implantable system or device can partially or completely(e.g., at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, up toand including 100% of the silk matrix) degrade within at least about 1week, at least about 2 weeks, at least about 3 weeks, at least about 4weeks, at least about 5 weeks, at least about 6 weeks, at least about 7weeks, at least about 8 weeks or more. In some embodiments, the silkmatrix within the silk-based implantable system or device can partiallyor completely (e.g., at least about 10%, at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, up to and including 100% of the silk matrix) degrade withinat least about 1 month, at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 7 months, at least about 8 months or more

The electrical component or electrically-conducting material in asilk-based implantable system or device can have a conductivity thatallows recording or detection of extracellular field potentials of atleast one cell or a cluster of cells. For example, the extracellularfield potentials are generally on the order of 100 μV for singleneurons, or ˜1-˜10 mV for neuronal clusters. In some embodiments, theelectrical component or electrically-conducting material in a silk-basedimplantable system or device can have a conductivity that allowsdetection of extracellular field potentials ranging from about 1 μV toabout 100 mV, from about 10 g±V to about 50 mV, from about 50 μV toabout 20 mV.

Stated another way, the electrical component or electrically-conductingmaterial present in a silk-based implantable system or device can have aresistivity comparable to (e.g., at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95% or more of)that of a metal, such as gold and platinum, typically used inmicroelectrodes. In some embodiments, the electrical component orelectrically-conducting material present in a silk-based implantablesystem or device can have a resistivity approaching on the order of1-500 nΩ·m, or 20-100 nΩ·m. From a device perspective, in someembodiments, the impedance of a silk-based implantable system or device(e.g., a silk-based electrode) can be in the range of about 0.5 kΩ toabout 50 MΩ, about 5 kΩ to about 10 MΩ, or about 50 kΩ to about 1 MΩ ata frequency of about 0.1 kHz to about 10 kHz, or about 0.5 kHz to about5 kHz in a physiologic environment. In some embodiments, the impedanceof a silk-based implantable system or device (e.g., a silk-basedelectrode) can be in the range of about 50 kΩ to about 1 MΩ at afrequency of about 1 kHz in a physiologic environment. In someembodiments, the electrical characteristics of the silk-based electrodecan be comparable to (e.g., at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95% or more of)existing brain penetrating electrodes.

The electrically-conducting component can include any material that canconduct electricity and/or is commonly used as an electrode contact.Examples of an electrically-conducting component can include, but arenot limited to a metal such as a transition metal and/or a noble metal.An exemplary transition metal for use in an electrically-conductingcomponent can include, without limitations, silicon and/or copper.Exemplary noble metals for use in an electrically-conducting componentcan include, but are not limited to, gold, platinum, titanium, any noblemetal commonly used as an electronic, and any combinations thereof.

In such embodiments, the electrically-conducting component can beincorporated in the silk-based implantable system or device by any knownmethods in the art. In some embodiments, such electrically-conductingcomponent can be deposited on a surface of the silk matrix or silk bodyat a desired position. In some embodiments, the electrically-conductingcomponent can be patterned on a surface of the silk matrix or silk bodyat a desired position using the patterning method as shown in FIG. 21.See details of the patterning method on Kim, D. H., et al “Siliconelectronics on silk as a path to resorbable implantable devices.” (2009)Applied Physics Letters 95(13):133701; and Kim, D. H. et al.“Dissolvable films of silk fibroin for ultrathin conformal biointegratedelectronics.” (2010) Nature Materials 9(6):511-517. These reportsindicate that silicon transistors can be fabricated on resorbable silkfilms for brain recordings on cats, with no inflammatory response invivo.

In some embodiments, biodegradable electronics, e.g., natural organicsemiconductors such as melanin and carotenoid families of chemicals(with semiconductor properties) can be used in place of conventionalelectronic components, to form a completely biodegradable implantablesystem or device.

In some embodiments, the electrically-conducting component can includesilk. While silk is inherently an insulator as a highly hydrophobicpolymer with low water content, silk can be modified to become aconductive material. For example, various processing of silk can turnsilk to become a piezoelectric material. Methods for producing apiezoelectric silk material are described in International Patent Appl.No. WO/2012/047682 entitled “Silk-based piezoelectric materials” filedSep. 27, 2011.

Additionally or alternatively, silk can be doped with a conductivematerial to generate a conductive composite material. Examples of aconductive material can include, but are not limited to, a metalparticle (e.g., a gold nanoparticle), carbon nanotube, graphene, aconducting polymer, and any combinations thereof. In one embodiment,silk can be doped with conductive gold nanoparticles. See, e.g., Tao H.et al. “Gold nanoparticle-doped biocompatible silk films as a path toimplantable thermo-electrically wireless powering devices” (2010) Appl.Phys. Lett. 97, 123702.

In another embodiment, silk can be doped with a conducting polymer.Exemplary conducting polymers for use as a dopant in silk can include,but are not limited to, polyethylenedioxythiophene (PEDOT),polypyrrole-based conductive polymer, copolymers of thiophenes andpolypyrroles, copolymers of poly-lactide and polyaniline, or anycombinations thereof. Conducting polymers have been reported for in vivouse, with low toxicity and good tissue compatibility. In someembodiments, a conducting polymer can be brittle, a property which canbe improved by blending with silk. It has been previously reported thatPEDOT can be used as a biocompatible coating for implantable electrodes,and that PEDOT can be polymerized around living cells. Alternatively,degradable/erodable conductive polymers can be blended with silk,including, but not limited to, erodible polypyrrole based conductivepolymer, degradable conductive copolymers of thiophenes and polypyrroleslinked by degradable ester linkages or degradable conducting copolymersconsisting of poly-lactide and polyaniline. In such embodiments, using adegradable/erodible conductive polymer as a dopant in silk can allow forthe synthesis of a fully resorbable conducting silk composite material.

The amount of a dopant present in a silk matrix body can vary withdesired conductivity and/or types and conductivity of dopants. In someembodiments, the dopant can be present in a silk matrix or silk body inan amount of about 0.01% w/w to about 50% w/w, about 0.05% w/w to about30% w/w, about 0.1% w/w to about 25% w/v.

In some embodiments, the electrically-conducting component can includesilk modified to conduct electricity (e.g., having a conductivitydefined herein, or having a resistivity defined herein). To make silkbecome conductive via growth or interfaces with conducting polymers, anexemplary approach of modifying the silk polymer itself to conductelectricity can involve surface functionalization of the silk to modifythe high content of tyrosines to sulfate groups. In some embodiments,the surface functionalization can be performed using diazonium couplingreactions (See, e.g., Murphy, A R. et al. “Modification of silk usingdiazonium coupling chemistry and the effects on hMSC proliferation anddifferentiation.” (2008) Biomaterials 29:2829-2838; and U.S. App. No. US2009/0232963 entitled “Diazonium salt modification of silk polymer”filed Aug. 15, 2008, the content of which is incorporated herein byreference). The modified tyrosines can be used as anchoring sites towhere the one or more conducting polymers can be polymerized forenhanced electronic interfaces.

Silk can stabilize labile substances including, but not limited to,enzymes, antibiotics, vaccines and small molecules, even at temperaturesat 60° C. over extended time frames, e.g., a period of at least 24 hoursor longer, such as for days, months or years. Without wishing to bebound by theory, the unique nano-domain structures in silk, along withthe high hydrophobic content of the assembled material, can provide asuitable environment to maintain function of otherwise labile materialsentrained in silk devices. Further, the entrapped biological componentscan retain function and the release profile can be controlled based onthe crystalline state of the silk matrix. See, e.g., Guziewicz, N., etal. “Lyophilized silk fibroin hydrogels for the sustained local deliveryof therapeutic monoclonal antibodies.” Biomaterials (2011) April;32(10):2642-50; Lu, Q., et al. “Stabilization and release of enzymesfrom silk films” Macromolecular Bioscience (2010) 10(4):359-368; Lu, S.,et al. “Stabilization of enzymes in silk films.” Biomacromolecules(2009) 10(5):1032-1042; Pritchard, E. M., “Incorpoatin of proteinaseinhibitors into silk-based delivery devices for enhanced control ofdegradation and drug release.” Biomaterials (2011) 32(3): 909-918;Pritchard, E. et al. “Silk fibroin encapsulated powder reservoirs forsustained release of adenosine.” (2010) J. Controlled Release144(2):159-167; Szybala, C. et al. “Antiepileptic effects ofsilk-polymer based adenosine release in kindled rats.” ExperimentalNeurology. (2009) 219(1):126-135; Witz, A. et al. “Silk polymer-basedadenosine release: therapeutic potential for epilepsy.” Biomaterials(2008) 29:3609-3616; Wang, X. et al. “Controlled release from multilayersilk biomaterial coatings to modulate vascular cell responses.”Biomaterials (2008) 29(7):894-903; Wang, X. et al. “Nanolayerbiomaterial coatings of silk fibroin for controlled release.” J.Controlled Release (2007) 121(3):190-199; and Wang, X. et al. “Silkmicrospheres for encapsulation and controlled release.” (2007) J.Controlled Release. 117:360-370.

Accordingly, in some embodiments, the silk matrix and/or silk body ofthe silk-based electrode can comprise one or more active agents(including, but not limited to one or more therapeutic agents). Theactive agent can be included in the silk solution prior to forming thesilk matrix and/or silk body, or can be coated on a surface of theformed silk matrix and/or silk body, or can be distributed or dispersedinto the formed silk matrix and/or silk body by diffusion. Methods forincorporating an active agent into the silk matrix and/or silk body ofthe silk matrix are described later herein.

In such embodiments, the amount of any active agent loaded into the silkmatrix or silk body is effective for producing a therapeutic effect in asubject for a certain period of time, e.g., for at least about 3 days,at least about 1 week, at least about 2 weeks, at least about 3 weeks,at least about 4 weeks, at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 9 months, at least about 12 months or longer.

As used herein, the term “therapeutic effect” refers to reducing atleast one adverse effect associated with implantation of a device into atissue, and/or at least one symptom associated with a disease ordisorder to be treated with the device. For example, for a silk-basedneuroprosthetic device implanted in a brain tissue, the active agentloaded therein is effective for reducing gliosis or scar formationaround the implanted neuroprosthetic device by at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90% or more, as compared to theabsence of the active agent. Alternatively or additionally, the activeagent loaded therein is effective for reducing at least one symptomassociated with a disease or disorder to be treated (or improvingneurological function) by at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90% or more, as compared to the absence of theactive agent.

Accordingly, in some embodiments, the active agent can be present in anamount about 0.01% (w/w) to about 90% (w/w) of the total weight (i.e.,the combined weight of the silk matrix and the therapeutic agent), forexample, including, about 0.01% (w/w) to about 70% (w/w), about 0.1%(w/w) to about 50% (w/w), about 1% (w/w) to about 30% (w/w), about 5%(w/w) to about 25% (w/w), or about 7.5% (w/w) to about 20 (w/w) of thetotal weight. In some embodiments, the therapeutic agent can be presentin a silk matrix in an amount of about 0.5% (w/w) to about 20% (w/w) ofthe total weight. In some embodiments, the therapeutic agent can bepresent in a silk matrix in an amount of about 2% (w/w) to about 20%(w/w) of the total weight. In one embodiment, the therapeutic agent canbe present in a silk matrix in an amount of about 1% (w/w) to about 20%(w/w) of the total weight. In one embodiment, the therapeutic agent canbe present in a silk matrix in an amount of about 0.1% (w/w) to 5% (w/w)of the total weight.

Silk can stabilize labile active agent including, but not limited to,enzymes, antibiotics, vaccines and small molecules, even at temperaturesat 60° C. over extended time frames, e.g., a period of at least 24 hoursor longer, such as for days, months or years. Without wishing to bebound by theory, the unique nano-domain structures in silk, along withthe high hydrophobic content of the assembled material, can provide asuitable environment to maintain function of otherwise labile materialsentrained in silk devices. Further, the entrapped biological componentscan retain function and the release profile can be controlled based onthe crystalline state of the silk matrix.

In some embodiments, the silk-based electrode can have a sharpened tipor tapered tip at an end for facilitating insertion or penetration intoa target tissue. The sharpened tip or tapered tip can have across-sectional dimension of about 1 nm to about 500 μm, about 10 nm toabout 250 μm, about 50 nm to about 125 μm, about 100 nm to about 100 μm,about 150 nm to about 50 μm, about 200 nm to about 10 μm, or about 300nm to about 5 μm. In one embodiment, one end of the silk-based electrodecan have a needle-like shape.

In some embodiments, a plurality of (e.g., at least 2 including 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more)silk-based electrodes described herein can be arranged into an array toform a microelectrode array, which can provide distributed and controlof recordings and/or electrical stimulation over a larger tissue area(e.g., brain tissue area).

Without wishing to be bound, some embodiments of the silk-basedelectrode described herein can be flexible electrodes (e.g., anelectrode that is not stiff enough to penetrate a target tissue, e.g., abrain tissue). For example, the silk tube of the silk-based electrodecan be formed from a low-concentration silk solution (e.g., less thanabout 10% w/v). In those embodiments, the flexible silk-based electrodecan be further treated to form a silk-based implantable system describedherein. For example, at least a portion of the flexible silk-basedelectrode described herein can be in contact or coated with a silkmatrix described herein. The silk matrix can then provide the flexiblesilk-based electrode with sufficient stiffness to penetrate a targettissue and become compliant upon the penetration.

Silk Fibroin and Silk Solution for Making the Systems and/or DevicesDescribed Herein

Silk fibroin protein have unique chemical and physical properties, e.g.,tunable degradation rates, controllable crystallinity due to hydrophobicbeta-sheet segments—desirable diffusion barriers for entrapped drugmolecules, an amino acidic nature that provides an inertmicroenvironment for drug encapsulation, as well as an aqueous-basedmaterial processing that is favorable for sensitive drug molecules.Silk-based biomaterials have been previously reported for theirbiocompatibility and biosafety for various in vivo applications, whichis comparable with or superior to other biodegradable materials, such ascollagen, hyaluronic acids, poly-lactic-co-glycolic acid (PLGA).

Silk is both biocompatible and biodegradable (31,32), with tunablemechanical properties and resorption rates based on controlledcrystallization of the protein (33). Silk can be processed into diversematerial formats including, without limitations, fibers, films, gels andsponges using only water as a solvent and under ambient conditions (34),which is conducive to the encapsulation of sensitive therapeutics.

The utility of silk as a biomaterial for applications in the nervoussystem has been previously reported. For example, silk-based nerveguides can be used as scaffolds for regeneration in the peripheralnervous system (35-40). However, these silk-based nerve guides areplaced into the peripheral nervous system surgically, and thus they donot rely on the silk mechanics to penetrate tissues. Silk has also beenreported to support and direct the growth of various cell types from thecentral nervous system (41, 42). In the brain, specifically, silk hasbeen utilized to deliver adenosine to reduce seizures in epileptic rats(43) and as a vehicle for enhancing conformal contact between a planarelectrode array and the brain's surface (44). In addition, silk can beutilized to delivery a drug to modulate an immune response to silkmatrix (e.g., silk films) at the brain surface.

As used herein, the term “silk fibroin” includes silkworm fibroin andinsect or spider silk protein. See e.g., Lucas et al., 13 Adv. ProteinChem. 107 (1958). Any type of silk fibroin can be used according toaspects provided herein. Silk fibroin produced by silkworms, such asBombyx mori, is the most common and represents an earth-friendly,renewable resource. For instance, silk fibroin used in a silk fibroinfiber can be attained by extracting sericin from the cocoons of B. mori.Organic silkworm cocoons are also commercially available. There are manydifferent silks, however, including spider silk (e.g., obtained fromNephila clavipes), transgenic silks, genetically engineered silks, suchas silks from bacteria, yeast, mammalian cells, transgenic animals, ortransgenic plants (see, e.g., WO 97/08315; U.S. Pat. No. 5,245,012), andvariants thereof, that can be used. In some embodiments, silk fibroincan be derived from other sources such as spiders, other silkworms,bees, and bioengineered variants thereof. In some embodiments, silkfibroin can be extracted from a gland of silkworm or transgenicsilkworms (see, e.g., WO 2007/098951).

Silk fiber generated from Bombyx mori silkworms generally have a tensilestrength of 500 MPa and some recombinant spider silks can have achievedtenacity (maximum fiber stress) of 508 MPa, close to properties fornative N. clavipes dragline silk (740-1200 MPa). Different processing ofsilk solution can improve the mechanical property of the silk fiber. Byway of example only, silk solution stored at various temperatures canallow mechanical drawing to produce outstanding mechanical properties.Some of these materials exhibited strong and tough fibers such astemperature-processed fibers (e.g., produced by a molding/freezingmethod, e.g., as described in the Prov. Appl. No. 61/477,486 filed Apr.20, 2012) with a diameter of approximately 0.42 mm and a modulus up to5,900 MPa. In some embodiments, the temperature-processed fibers can bestiff enough for insertion through the dura. For example, thetemperature-processed fibers remain undeformed (e.g., substantiallystraight) during insertion through the dura.

The silk fibroin solution can be prepared by any conventional methodknown to one skilled in the art. For example, B. mori cocoons are boiledfor about 30 minutes in an aqueous solution. In one embodiment, theaqueous solution is about 0.02M Na₂CO₃. The cocoons are rinsed, forexample, with water to extract the sericin proteins and the extractedsilk is dissolved in an aqueous salt solution. Salts useful for thispurpose include lithium bromide, lithium thiocyanate, calcium nitrate orother chemicals capable of solubilizing silk. In some embodiments, theextracted silk is dissolved in about 8M-12 M LiBr solution. The salt isconsequently removed using, for example, dialysis.

If necessary, the solution can then be concentrated using, for example,dialysis against a hygroscopic polymer, for example, PEG, a polyethyleneoxide, amylose or sericin. In some embodiments, the PEG is of amolecular weight of 8,000-10,000 g/mol and has a concentration of25%-50%. A slide-a-lyzer dialysis cassette (Pierce, MW CO 3500) can beused. However, any dialysis system may be used. The dialysis can beperformed for a time period sufficient to result in a final stockconcentration of aqueous silk solution between about 6% (w/v)-about 30%(w/v). In one embodiment, the dialysis can be performed for a timeperiod sufficient to result in a final stock concentration of aqueoussilk solution of about 15% (w/v). In most cases dialysis for 2-12 hoursis sufficient. See, for example, International Application No. WO2005/012606, the content of which is incorporated herein by reference.

Alternatively, the silk fibroin solution can be produced using organicsolvents. Such methods have been described, for example, in Li, M., etal., J. Appl. Poly Sci. 2001, 79, 2192-2199; Min, S., et al. Sen'IGakkaishi 1997, 54, 85-92; Nazarov, R. et al., Biomacromolecules 2004May-June; 5(3):718-26. For example, an exemplary organic solvent thatcan be used to produce a silk solution includes, but is not limited to,hexafluoroisopropanol.

A silk solution for use in making the silk matrix or silk body of theimplantable system and/or device described herein can comprise fibroinat any concentration, depending on desired characteristics of the silkmatrix or silk body, e.g., drug release profile and/or its solubility,e.g., in water. In some embodiments, the silk solution can comprise silkfibroin at a concentration of about 0.1% (w/v) to about 50% (w/v), about1% (w/v) to about 40% (w/v), about 6% (w/v) to about 30% (w/v), or about7% (w/v) to about 20% (w/v). In some embodiments, the silk solution cancomprise silk fibroin at a concentration of about 10% (w/v) to about 30%(w/v). In some embodiments, the silk solution can comprise silk fibroinat a concentration greater than 5% (w/v), greater than 10% (w/v), orgreater than 15% (w/v). Generally, higher silk concentration can resultin a silk matrix or silk body with a higher tensile modulus.

In various embodiments, the silk fibroin can be modified for differentapplications and/or desired mechanical or chemical properties (e.g., tofacilitate formation of a gradient of a therapeutic agent in silkfibroin matrices). One of skill in the art can select appropriatemethods to modify silk fibroins, e.g., depending on the side groups ofthe silk fibroins, desired reactivity of the silk fibroin and/or desiredcharge density on the silk fibroin. In one embodiment, modification ofsilk fibroin can use the amino acid side chain chemistry, such aschemical modifications through covalent bonding, or modificationsthrough charge-charge interaction. Exemplary chemical modificationmethods include, but are not limited to, carbodiimide coupling reaction(see, e.g. U.S. Patent Application. No. US 2007/0212730), diazoniumcoupling reaction (see, e.g., U.S. Patent Application No. US2009/0232963), avidin-biotin interaction (see, e.g., InternationalApplication No.: WO 2011/011347) and pegylation with a chemically activeor activated derivatives of the PEG polymer (see, e.g., InternationalApplication No. WO 2010/057142). Silk fibroin can also be modifiedthrough gene modification to alter functionalities of the silk protein(see, e.g., International Application No. WO 2011/006133). For instance,the silk fibroin can be genetically modified, which can provide forfurther modification of the silk such as the inclusion of a fusionpolypeptide comprising a fibrous protein domain and a mineralizationdomain, which can be used to form an organic-inorganic composite. See WO2006/076711. In some embodiments, the silk fibroin can be geneticallymodified to be fused with a protein, e.g., a therapeutic protein.Additionally, the silk fibroin matrix can be combined with a chemical,such as glycerol, that, e.g., affects flexibility and/or solubility ofthe matrix. See, e.g., WO 2010/042798, Modified Silk films ContainingGlycerol.

In some embodiments, the silk solution for use in making the silk matrixor silk body of the implantable system and/or device described hereincan further comprise one or more (e.g., one, two, three, four, five ormore) additives, e.g., for various desired properties and/orapplications. Exemplary additives can include, but are not limited to, abiopolymer, a porogen (e.g., a salt or polymeric particle), a magneticparticle, a plasmonic particle, a metamaterial, an excipient, aplasticizer (e.g., glycerol, polyvinyl alcohol, collagen, gelatin,alginate, chitosan, hyaluronic acid, polyethylene glycol, polyethyleneoxide, and any combinations thereof), a detection label, and anycombinations thereof. The additive(s) can be present in the silksolution at any ratio. For example, the weight ratio of the additive tosilk in the silk solution can range from about 1:1000 to about 1000:1,or from about 1:100 to about 100:1, or from about 1:10 to about 10:1. Insome embodiments, total amount of additives in the solution can be fromabout 0.1 wt % to about 70 wt %, from about 5 wt % to about 60 wt %,from about 10 wt % to about 50 wt %, from about 15 wt % to about 45 wt%, or from about 20 wt % to about 40 wt %, of the total silk fibroin inthe solution.

In some embodiments, at least one additive added into the silk solutionfor preparing a silk matrix or silk body of the implantable system caninclude one or more (e.g., one, two, three, four, five or more)biopolymers and/or biocompatible polymers. Exemplary biopolymers and/orbiocompatible polymers include, but are not limited to, a poly-lacticacid (PLA), poly-glycolic acid (PGA), poly-lactide-co-glycolide (PLGA),polyesters, poly(ortho ester), poly(phosphazine), poly(phosphate ester),polycaprolactone, gelatin, collagen, fibronectin, keratin, polyasparticacid, alginate, chitosan, chitin, hyaluronic acid, pectin,polyhydroxyalkanoates, dextrans, and polyanhydrides, polyethylene oxide(PEO), poly(ethylene glycol) (PEG), triblock copolymers, polylysine,alginate, polyaspartic acid, sugar, tyrosine-based polymers, polyvinylacetate, cellulose, any derivatives thereof and any combinationsthereof. Other exemplary biocompatible polymers amenable to useaccording to the present disclosure include those described for examplein U.S. Pat. Nos. 6,302,848; 6,395,734; 6,127,143; 5,263,992; 6,379,690;5,015,476; 4,806,355; 6,372,244; 6,310,188; 5,093,489; 387,413;6,325,810; 6,337,198; 6,267,776; 5,576,881; 6,245,537; 5,902,800; and5,270,419, content of all of which is incorporated herein by reference.

In some embodiments, the silk solution can comprise particles formechanical reinforcement when making a silk matrix or silk body. In someembodiments, the particles can include silk particles. For example,ultrafine silk particles can be added into the silk solution for forminga silk matrix or silk body, thus controlling mechanical features.Without wishing to be bound by theory, the silk particles can retaintheir crystallinity and are useful for matrix/body reinforcement due tointerfacial compatibility, resulting in significant improvements inmechanical properties. The presence of the particles can increase thewet compression modulus and the yield strength of the silk matrix orsilk body about two orders of magnitude or higher in comparison to thescaffolds without the particles. Without wishing to be bound by theory,this significant increase can be due to the high interfacial cohesionbetween the matrix and the particle reinforcements due to partialsolubility of crystalline silk particles in a solvent (e.g.,aqueous-based solvent or organic solvent such as HFIP). See, e.g.,Rajkhowa, R., et al. “Reinforcing silk scaffolds with silk particles.”Macromolecular Bioscience. (2010) 10(6): 599-611.

Any embodiment of the silk solution described herein for preparing thesilk matrix and/or silk body of the implantable system and/or devicedescribed herein can also be used to fill the silk reservoir of thesilk-based implantable device described herein. For example, the silksolution present in the silk reservoir of the silk-based implantabledevice described herein can also include at least one active agent,and/or at least one additive described herein. However, in someembodiments, the concentration of the silk solution stored in the silkreservoir can be lower than the concentration of the silk solution usedto make the silk body. In some embodiments, the silk solution stored inthe silk reservoir need not any particles for mechanical reinforcement.In some embodiments, the silk solution stored in the silk reservoir canhave a concentration that would allow the silk solution remaining in asolution or fluid state after implantation such that it can bedischarged through the through holes on an electrode surface to form asilk coating thereon. However, the concentration of the silk solutionstored in the silk reservoir should not be so low that it can compromisethe durability of the silk coating.

Exemplary Therapeutic Agents and Amounts Thereof in a Silk Matrix orSilk Body

Depending on various applications of the implantable systems and/ordevices described herein, different types of the active agent can bepresent in the silk matrix or silk body, e.g., by encapsulation and/orcoating. Without wishing to be bound, for example, the silk matrix orsilk body can comprise one or more active agents, including, but notlimited to, therapeutic agents, imaging agents or any combinationsthereof.

In some embodiments, one or more imaging agents can be included in asilk matrix or silk body. Examples of imaging agents can include, butare not limited to, dyes, fluorescent agents, radiological imagingagents, any art-recognized contrast agents for imaging tissues and/ororgans, and any combinations thereof. Fluorescent agents are well knownin the art. Examples of fluorescent agents can include, but are notlimited to, fluoresceinisothiocyanato-dextran (FITC-dextran), rutheniumbased dye, or platinum porphyrin, or a mixture thereof.

As used herein, the term “therapeutic agent” means a molecule, group ofmolecules, complex or substance administered to an organism fordiagnostic, therapeutic, preventative medical, or veterinary purposes.As used herein, the term “therapeutic agent” includes a “drug” or a“vaccine.” This term include externally and internally administeredtopical, localized and systemic human and animal pharmaceuticals,treatments, remedies, nutraceuticals, cosmeceuticals, biologicals,devices, diagnostics and contraceptives, including preparations usefulin clinical and veterinary screening, prevention, prophylaxis, healing,wellness, detection, imaging, diagnosis, therapy, surgery, monitoring,cosmetics, prosthetics, forensics and the like. This term can also beused in reference to agriceutical, workplace, military, industrial andenvironmental therapeutics or remedies comprising selected molecules orselected nucleic acid sequences capable of recognizing cellularreceptors, membrane receptors, hormone receptors, therapeutic receptors,microbes, viruses or selected targets comprising or capable ofcontacting plants, animals and/or humans. This term can alsospecifically include nucleic acids and compounds comprising nucleicacids that produce a therapeutic effect, for example deoxyribonucleicacid (DNA), ribonucleic acid (RNA), or mixtures or combinations thereof.

The term “therapeutic agent” also includes an agent that is capable ofproviding a local or systemic biological, physiological, or therapeuticeffect in the biological system to which it is applied. For example, thetherapeutic agent can act to control infection or inflammation, enhancecell growth and tissue regeneration, suppress cell proliferation,control tumor growth, act as an analgesic, promote anti-cell attachment,and enhance bone growth, among other functions. Other suitabletherapeutic agents can include anti-viral agents, hormones, antibodies,or therapeutic proteins. Other therapeutic agents include prodrugs,which are agents that are not biologically active when administered but,upon administration to a subject are converted to biologically activeagents through metabolism or some other mechanism. Additionally, a silkmatrix can contain combinations of two or more therapeutic agents.

A therapeutic agent can include a wide variety of different compounds,including chemical compounds and mixtures of chemical compounds, e.g.,small organic or inorganic molecules; saccharines; oligosaccharides;polysaccharides; biological macromolecules, e.g., peptides, proteins,and peptide analogs and derivatives; peptidomimetics; antibodies andantigen binding fragments thereof; nucleic acids; nucleic acid analogsand derivatives; an extract made from biological materials such asbacteria, plants, fungi, or animal cells; animal tissues; naturallyoccurring or synthetic compositions; and any combinations thereof. Insome embodiments, the therapeutic agent is a small molecule.

As used herein, the term “small molecule” can refer to compounds thatare “natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” compounds. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight of less than 5000 Daltons (5 kDa), preferablyless than 3 kDa, still more preferably less than 2 kDa, and mostpreferably less than 1 kDa. In some cases it is preferred that a smallmolecule have a molecular weight equal to or less than 700 Daltons.

Exemplary therapeutic agents include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; Physicians DeskReference, 50^(th) Edition, 1997, Oradell New Jersey, Medical EconomicsCo.; Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990, the complete contents of all of which areincorporated herein by reference.

Therapeutic agents include the herein disclosed categories and specificexamples. It is not intended that the category be limited by thespecific examples. Those of ordinary skill in the art will recognizealso numerous other compounds that fall within the categories and thatare useful according to the present disclosure. Examples include aradiosensitizer, a steroid, a xanthine, a beta-2-agonist bronchodilator,an anti-inflammatory agent, an analgesic agent, a calcium antagonist, anangiotensin-converting enzyme inhibitors, a beta-blocker, a centrallyactive alpha-agonist, an alpha-1-antagonist, ananticholinergic/antispasmodic agent, a vasopressin analogue, anantiarrhythmic agent, an anti-parkinsonian agent, anantiangina/antihypertensive agent, an anticoagulant agent, anantiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, abiopolymeric agent, an antineoplastic agent, a laxative, anantidiarrheal agent, an antimicrobial agent, an antifingal agent, avaccine, a protein, or a nucleic acid. In a further aspect, thepharmaceutically active agent can be coumarin, albumin, steroids such asbetamethasone, dexamethasone, methylprednisolone, prednisolone,prednisone, triamcinolone, budesonide, hydrocortisone, andpharmaceutically acceptable hydrocortisone derivatives; xanthines suchas theophylline and doxophylline; beta-2-agonist bronchodilators such assalbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol;antiinflammatory agents, including antiasthmatic anti-inflammatoryagents, antiarthritis antiinflammatory agents, and non-steroidalantiinflammatory agents, examples of which include but are not limitedto sulfides, mesalamine, budesonide, salazopyrin, diclofenac,pharmaceutically acceptable diclofenac salts, nimesulide, naproxene,acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agentssuch as salicylates; calcium channel blockers such as nifedipine,amlodipine, and nicardipine; angiotensin-converting enzyme inhibitorssuch as captopril, benazepril hydrochloride, fosinopril sodium,trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride,and moexipril hydrochloride; beta-blockers (i.e., beta adrenergicblocking agents) such as sotalol hydrochloride, timolol maleate, esmololhydrochloride, carteolol, propanolol hydrochloride, betaxololhydrochloride, penbutolol sulfate, metoprolol tartrate, metoprololsuccinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprololfumarate; centrally active alpha-2-agonists such as clonidine;alpha-1-antagonists such as doxazosin and prazosin;anticholinergic/antispasmodic agents such as dicyclomine hydrochloride,scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate,and oxybutynin; vasopressin analogues such as vasopressin anddesmopressin; antiarrhythmic agents such as quinidine, lidocaine,tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamilhydrochloride, propafenone hydrochloride, flecainide acetate,procainamide hydrochloride, moricizine hydrochloride, and disopyramidephosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa,selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, andbromocryptine; antiangina agents and antihypertensive agents such asisosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol andverapamil; anticoagulant and antiplatelet agents such as Coumadin,warfarin, acetylsalicylic acid, and ticlopidine; sedatives such asbenzodiazapines and barbiturates; ansiolytic agents such as lorazepam,bromazepam, and diazepam; peptidic and biopolymeric agents such ascalcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin,insulin, somatostatin, protirelin, interferon, desmopressin,somatotropin, thymopentin, pidotimod, erythropoietin, interleukins,melatonin, granulocyte/macrophage-CSF, and heparin; antineoplasticagents such as etoposide, etoposide phosphate, cyclophosphamide,methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin,hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase,altretamine, mitotane, and procarbazine hydrochloride; laxatives such assenna concentrate, casanthranol, bisacodyl, and sodium picosulphate;antidiarrheal agents such as difenoxine hydrochloride, loperamidehydrochloride, furazolidone, diphenoxylate hdyrochloride, andmicroorganisms; vaccines such as bacterial and viral vaccines;antimicrobial agents such as penicillins, cephalosporins, andmacrolides, antifungal agents such as imidazolic and triazolicderivatives; and nucleic acids such as DNA sequences encoding forbiological proteins, and antisense oligonucleotides.

In some embodiments, the active agent to be included in a silk matrix orsilk body can include an agent (including a therapeutic agent) that canpromote tissue growth, reduce scar formation, control inflammation at apenetration site (e.g., reducing inflammation at a penetration site), orany combinations thereof. Examples of anti-inflammatory agents and/orscar-reducing agents can include, but are not limited to, dexamethasone(23-26), alpha-MSH (27), cell cycle inhibitor flavopiridol (28), neuraladhesion molecule L1 (29), and any combinations thereof.

In some embodiments, the active agent to be included in a silk matrix orsilk body can include at least one, at least two, at least three, atleast four, at least five or more gliosis-modulating agents (e.g., anagent that can reduce or inhibit proliferation of astrocytes in damagedareas of the central nervous system). An exemplary gliosis-modulatingagent can include cytarabine (or Arabinofuranosyl Cytidine).Additionally or alternatively, the active agent to be included in a silkmatrix or silk body can include at least one, at least two, at leastthree, at least four, at least five or more antibiotics. In someembodiments, the antibiotics can include, but are not limited to,penicillin.

As noted above, any therapeutic agent can be included in a silk matrixor silk body, e.g., by encapsulation and/or coating. In someembodiments, it is desirable to include in a silk matrix material topromote the growth of the agent (for biological agents), promote thefunctionality of the agent after it is released from the encapsulation,or increase the agent's ability to survive or retain its efficacy duringthe encapsulation period. Materials known to promote cell growth includecell growth media, such as Dulbecco's Modified Eagle Medium (DMEM),fetal bovine serum (FBS), non-essential amino acids and antibiotics, andgrowth and morphogen factors such as basic fibroblast growth factor(bFGF), transforming growth factors (TGFs), Vascular endothelial growthfactor (VEGF), insulin-like growth factor (IGF-I), bone morphogeneticgrowth factors (BMPs), nerve growth factors and related proteins.

Additional options for delivery via the silk matrix or silk bodydescribed herein can include DNA, siRNA, antisense, plasmids, liposomesand related systems for delivery of genetic materials; antibodies andantigen binding fragment thereof; peptides and proteins to activecellular signaling cascades; peptides and proteins to promotemineralization or related events from cells; adhesion peptides andproteins to improve gel-tissue interfaces; antimicrobial peptides; andproteins and related compounds.

In some embodiments, the therapeutic agent(s) for use in the presentdisclosure include, but are not limited to, those requiring relativelyfrequent dosing. For example, those used in the treatment of chronicdisorders or conditions.

In some embodiments, the therapeutic agent is a cell, e.g. a biologicalcell. In such embodiments, the cells can be distributed within a silkmatrix by incubating the silk matrix in a cell suspension, where thecells can migrate from the suspension into the pores of the silk matrix.Cells amenable to be incorporated into the silk matrix include, but arenot limited to, stem cells (embryonic stem cells, mesenchymal stemcells, bone-marrow derived stem cells and hematopoietic stem cells),chrondrocytes progenitor cells, pancreatic progenitor cells, myoblasts,fibroblasts, keratinocytes, neuronal cells, glial cells, astrocytes,pre-adipocytes, adipocytes, vascular endothelial cells, hair follicularstem cells, endothelial progenitor cells, mesenchymal cells, neural stemcells and smooth muscle progenitor cells.

In some embodiments, the cell is a genetically modified cell. A cell canbe genetically modified to express and secrete a desired compound, e.g.a bioactive agent, a growth factor, differentiation factor, cytokines,and the like. Methods of genetically modifying cells for expressing andsecreting compounds of interest are known in the art and easilyadaptable by one of skill in the art.

Differentiated cells that have been reprogrammed into stem cells canalso be used. For example, human skin cells reprogrammed into embryonicstem cells by the transduction of Oct3/4, Sox2, c-Myc and Klf4 (JunyingYu, et. al., Science, 2007, 318, 1917-1920 and Takahashi K. et. al.,Cell, 2007, 131, 1-12).

Cells useful for incorporation into the silk matrix can come from anysource, for example human, rat or mouse. Human cells include, but arenot limited to, human cardiac myocytes-adult (HCMa), human dermalfibroblasts-fetal (HDF-f), human epidermal keratinocytes (HEK), humanmesenchymal stem cells-bone marrow, human umbilical mesenchymal stemcells, human hair follicular inner root sheath cells, human umbilicalvein endothelial cells (HUVEC), and human umbilical vein smooth musclecells (HUVSMC), human endothelial progenitor cells, human myoblasts,human capillary endothelial cells, and human neural stem cells.

Exemplary rat and mouse cells include, but not limited to, RN-h (ratneurons-hippocampal), RN-c (rat neurons-cortical), RA (rat astrocytes),rat dorsal root ganglion cells, rat neuroprogenitor cells, mouseembryonic stem cells (mESC) mouse neural precursor cells, mousepancreatic progenitor cells mouse mesenchymal cells and mouse endodermalcells.

In some embodiments, tissue culture cell lines can be used in the silkmatrix described herein. Examples of cell lines include, but are notlimited to, C166 cells (embryonic day 12 mouse yolk), C6 glioma Cellline, HL1 (cardiac muscle cell line), AML12 (nontransforminghepatocytes), HeLa cells (cervical cancer cell line) and Chinese HamsterOvary cells (CHO cells).

An ordinary skill artisan in the art can locate, isolate and expand suchcells. In addition, the basic principles of cell culture and methods oflocating, isolation and expansion and preparing cells for tissueengineering are described in “Culture of Cells for Tissue Engineering”Editor(s): Gordana Vunjak-Novakovic, R. Ian Freshney, 2006 John Wiley &Sons, Inc., and Heath C. A., Trends in Biotechnology, 2000, 18, 17-19,content of both of which is herein incorporated by reference in itsentirety.

Generally, any amount of the therapeutic agent can be dispersed orencapsulated in the silk matrix or silk body, depending on a number offactors, including, but not limited to, desirable release profile (e.g.,release rates and/or duration), properties (e.g., half-life and/ormolecular size) and/or potency of the therapeutic agent, severity of asubject's disease or disorder to be treated, desirable administrationschedule, loading capacity of the silk matrix, and any combinationsthereof. For example, in some embodiments, a therapeutic agent can bepresent in a silk matrix or silk body in an amount of about 1 ng toabout 100 mg, about 500 ng to about 90 mg, about 1 μg to about 75 mg,about 0.01 mg to about 50 mg, about 0.1 mg to about 50 mg, about 1 mg toabout 40 mg, about 5 mg to about 25 mg. In some embodiments, atherapeutic agent can be present in a silk matrix or silk body in anamount of about 0.01% (w/w) to about 90% (w/w) of the total weight(i.e., the combined weight of the silk matrix or silk body and thetherapeutic agent), for example, including, about 0.01% (w/w) to about70% (w/w), about 0.1% (w/w) to about 50% (w/w), about 1% (w/w) to about30% (w/w), about 5% (w/w) to about 25% (w/w), or about 7.5% (w/w) toabout 20 (w/w) of the total weight. In some embodiments, the therapeuticagent can be present in a silk matrix or silk body in an amount of about0.5% (w/w) to about 20% (w/w) of the total weight. In some embodiments,the therapeutic agent can be present in a silk matrix or silk body in anamount of about 2% (w/w) to about 20% (w/w) of the total weight. In oneembodiment, the therapeutic agent can be present in a silk matrix orsilk body in an amount of about 0.1% (w/w) to 5% (w/w) of the totalweight.

Without wishing to be bound by theory, the duration of a therapeuticeffect on a target site to be treated is generally correlated with howlong an amount of the therapeutic agent delivered to the target site canbe maintained at a therapeutically effective amount. Thus, in someembodiments, the implantable system and/or device described herein cancomprise a therapeutic agent dispersed or encapsulated in a silk matrix,wherein the therapeutic agent is present in an amount sufficient tomaintain a therapeutically effective amount thereof delivered to treat atarget site, upon implantation, over a specified period of time, e.g.,over more than 1 week, or more than 1 month.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent which is effective for producing abeneficial or desired clinical result in at least a sub-population ofcells in a subject at a reasonable benefit/risk ratio applicable to anymedical treatment. For example, a therapeutically effective amountdelivered to a target site is sufficient to, directly or indirectly,produce a statistically significant, measurable therapeutic effect asdefined herein. By way of example only, the therapeutically effectiveamount delivered to a target site for treatment is sufficient to reduceat least one symptom or marker associated with the disease or disorderto be treated (e.g., but not limited to, cancer such as brain cancer,cardiovascular diseases such as cardiac arrhythmia, neurodegenerativediseases such as Alzheimer's disease) by at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60% or higher, as compared to absence of the therapeuticagent. In some embodiments, the therapeutically effective amountdelivered to a target site for treatment is sufficient to reduce atleast one symptom or marker associated with the disease or disorder tobe treated (e.g., but not limited to, cancer such as brain cancer,cardiovascular diseases such as cardiac arrhythmia, neurodegenerativediseases such as Alzheimer's disease) by at least about 60%, at leastabout 70%, at least about 80% or higher, as compared to absence of thetherapeutic agent. In some embodiments, the therapeutically effectiveamount delivered to a target site is sufficient to reduce at least onesymptom or marker associated with the disease or disorder to be treated(e.g., but not limited to, cancer such as brain cancer, cardiovasculardiseases such as cardiac arrhythmia, neurodegenerative diseases such asAlzheimer's disease) by at least about 80%, at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, up to and including100%, as compared to absence of the therapeutic agent.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.Furthermore, therapeutically effective amounts will vary, as recognizedby those skilled in the art, depending on the specific disease treated,the route of administration, the excipient selected, and the possibilityof combination therapy. In some embodiments, the therapeuticallyeffective amount can be in a range between the ED50 and LD50 (a dose ofa therapeutic agent at which about 50% of subjects taking it arekilled). In some embodiments, the therapeutically effective amount canbe in a range between the ED50 (a dose of a therapeutic agent at which atherapeutic effect is detected in at least about 50% of subjects takingit) and the TD50 (a dose at which toxicity occurs at about 50% of thecases). In alternative embodiments, the therapeutically effective amountcan be an amount determined based on the current dosage regimen of thesame therapeutic agent administered in a non-silk matrix. For example,an upper limit of the therapeutically effective amount can be based on aconcentration or an amount of the therapeutic agent delivered to atarget site, on the day of administration with the current dosage of thetherapeutic agent in a non-silk matrix; while the lower limit of thetherapeutically effective amount can be based on a concentration or anamount of the therapeutic agent delivered to a target site, on the dayat which a fresh dosage of the therapeutic agent in a non-silk matrix isrequired.

As used herein, the term “maintain” is used in reference to sustaining aconcentration or an amount of a therapeutic agent delivered to a targetsite at least about or above the therapeutically effective amount over aspecified period of time. In some embodiments, the term “maintain” asused herein can refer to keeping the concentration or amount of atherapeutic agent at an essentially constant value over a specifiedperiod of time. In some embodiments, the term “maintain” as used hereincan refer to keeping the concentration or amount of a therapeutic agentwithin a range over a specified period of time. For example, theconcentration or amount of a therapeutic agent delivered to a targetsite can be maintained within a range between about the ED50 and aboutthe LD50 or between about the ED50 and about the TD50 over a specifiedperiod of time. In such embodiments, the concentration or amount of atherapeutic agent delivered to a target site can vary with time, but iskept within the therapeutically effective amount range for at least 90%of the specified period of time (e.g., at least about 95%, about 98%,about 99%, up to and including 100%, of the specified period of time).

In some embodiments, the therapeutic agent can be present in an amountsufficient to maintain a therapeutically effective amount thereofdelivered to a target site, upon implantation, over a period of morethan 1 week, including, e.g., at least about 2 weeks, at least about 3weeks, at least about 1 month, at least about 2 months, at least about 3months, at least about 6 months, at least about 12 months or longer.Such amounts of the therapeutic agent present in a silk matrix or silkbody can be generally smaller, e.g., at least about 10% smaller, thanthe amount of the therapeutic agent present in the current dosage of thetreatment regimen (i.e., without silk matrix) required for producingessentially the same therapeutic effect. Accordingly, a silk matrix orsilk body can comprise the therapeutic agent in an amount which is lessthan the amount recommended for one dosage of the therapeutic agent. Forexample, if the recommended dosage of the therapeutic agent is X amountthen the silk matrix or silk body can comprise a therapeutic agent in anamount of about 0.9×, about 0.8×, about 0.7×, about 0.6×, about 0.5×,about 0.4×, about 0.3×, about 0.2×, about 0.1× or less. Without wishingto be bound by a theory, this can allow administering a lower dosage ofthe therapeutic agent in a silk matrix or silk body to obtain atherapeutic effect which is similar to when a higher dosage isadministered without the silk matrix.

In some embodiments, an amount of the therapeutic agent dispersed orencapsulated in a silk matrix or silk body can be more than the amountgenerally recommended for one dosage of the same therapeutic agentadministered for a particular indication. Administration of atherapeutic agent in solution does not generally allow controlled andsustained release. Thus, release rate of a therapeutic agent in solutioncan generally create a higher initial burst and/or overall fasterrelease kinetics than that of the same amount of the therapeutic agentloaded in silk matrix or silk body. However, the silk matrix can act asa depot such that an amount of the therapeutic agent loaded in a silkmatrix or silk body can be higher than the amount generally recommendedfor one dosage of the same therapeutic agent and release the therapeuticagent over a period of time, thus providing a longer therapeutic effectwith lower frequency of administration. Accordingly, if the recommendeddosage of the therapeutic agent is X amount then the silk matrix canencapsulate a therapeutic agent in an amount of about 1.25×, about 1.5×,about 1.75×, about 2×, about 2.5×, about 3×, about 4×, about 5×, about6×, about 7×, about 8×, about 9×, about 10× or more. Without wishing tobe bound by a theory, this can allow administering the therapeutic agentin a silk matrix or silk body to obtain a therapeutic effect which issimilar to one obtained with multiple administration of the therapeuticagent administered without the silk matrix or silk body describedherein.

In some embodiments, an amount of the therapeutic agent encapsulated ordispersed in a dosage of the silk matrix or silk body can be essentiallythe same amount recommended for one dosage of the therapeutic agent. Forexample, if the recommended dosage of the therapeutic agent is X amount,then the silk-based composition can comprise about X amount of thetherapeutic agent. Without wishing to be bound by a theory, this canallow less frequent administration of the therapeutic agent to obtain atherapeutic effect over a longer period of time.

As used herein, the term “sustained delivery” refers to continualdelivery of a therapeutic agent in vivo or in vitro over a period oftime following administration. For example, sustained release can occurover a period of at least about 3 days, at least about a week, at leastabout two weeks, at least about three weeks, at least about four weeks,at least about 1 month, at least about 2 months, at least about 3months, at least about 4 months, at least about 5 months, at least about6 months, at least about 7 months, at least about 8 months, at leastabout 9 months, at least about 10 months, at least about 11 months, atleast about 12 months or longer. In some embodiments, the sustainedrelease can occur over a period of more than one month or longer. Insome embodiments, the sustained release can occur over a period of atleast about three months or longer. In some embodiments, the sustainedrelease can occur over a period of at least about six months or longer.In some embodiments, the sustained release can occur over a period of atleast about nine months or longer. In some embodiments, the sustainedrelease can occur over a period of at least about twelve months orlonger.

Sustained delivery of the therapeutic agent in vivo can be demonstratedby, for example, the continued therapeutic effect of the agent overtime. Alternatively, sustained delivery of the therapeutic agent can bedemonstrated by detecting the presence or level of the therapeutic agentin vivo over time. The release rate of a therapeutic agent can beadjusted by a number of factors such as silk matrix composition and/orconcentration, porous property of the silk matrix or silk body,molecular size of the therapeutic agent, and/or interaction of thetherapeutic agent with the silk matrix or silk body. For example, if thetherapeutic agent has a higher affinity with the silk matrix or silkbody, the release rate is usually slower than the one with a loweraffinity with the silk matrix or silk body. Additionally, when a silkmatrix or silk body has larger pores, the encapsulated therapeutic agentis generally released from the silk matrix or silk body faster than froma silk matrix or silk body with smaller pores.

In some embodiments, the therapeutic agent can be present in an amountto provide a release profile of the therapeutic agent from the silkmatrix or silk body such that the amount of the therapeutic agentdelivered to a target site is maintained within a therapeuticallyeffective amount range over a period of time. In some embodiments, thetherapeutic agent can be present in an amount to provide a releaseprofile of the therapeutic agent with release rates ranging from about0.01 ng/day to about 1000 mg/day, from about 0.1 ng/day to about 500mg/day, or from about 1 ng/day to about 250 mg/day over a period oftime. Without wishing to be bound by theory, upon implantation of theimplantable system and/or device described herein, there can be aninitial spike in the amount of the therapeutic agent released from thesilk matrix or silk body to a target site, and then the release rate ofthe therapeutic agent from the silk matrix or silk body can decreaseover a period of time. Thus, the therapeutic agent can be releasedinitially at a rate as high as mg/day, and later released in a slowerrate, e.g., in μg/day or ng/day. Accordingly, in some embodiments, thetherapeutic agent can be present in an amount to provide a releaseprofile such that daily release of the therapeutic agent can range fromabout 1 ng/day to about 1000 mg/day. For example, amount released can bein a range with a lower limit of from 1 to 1000 (e.g., every integerfrom 1 to 1000) and upper limit of from 1 to 1000 (e.g. every integerfrom 1 to 1000), wherein the lower and upper limit units can be selectedindependently from ng/day, μg/day, mg/day, or any combinations thereof.

In some embodiments, daily release can vary from about 1 rig/day toabout 10 mg/day, from about 0.25 rig/day to about 2.5 mg/day, or fromabout 0.5 rig/day to about 5 mg/day. In some embodiments, daily releaseof the therapeutic agent can range from about 100 ng/day to 1 mg/day,for example, or about 500 ng/day to 5 mg/day, or about 100 μg/day.

Stated another way, the therapeutic agent can be released from the silkmatrix or silk body at a rate such that at least about 5%, including,e.g., at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90% or more, of the therapeuticagent initially present in the silk matrix or silk body can be releasedover a period of about 3 days, about 1 week, about 10 days, about 20days, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, about 6 months, about 7 months, about 8 months, about 9months, about 10 months, about 11 months, about 12 months or longer. Insome embodiments, the therapeutic agent can be released from the silkmatrix or silk body at a rate such that about 5-30% of the therapeuticagent initially present in the silk matrix can be released over a periodof about 3-20 days. In some embodiments, the therapeutic agent can bereleased from the silk matrix or silk body at a rate such that about40-90% of the therapeutic agent initially present in the silk matrix orsilk body can be released over a period of about 3-30 days.

The release profiles of the therapeutic agent from the silk matrix orsilk body of the implantable system and/or device described herein canbe modulated by a number of factors such as amounts and/or molecularsize of the therapeutic agents loaded in a silk matrix or silk body,porosity of the silk matrix or silk body, amounts of silk fibroin in asilk matrix or silk body and/or contents of beta-sheet conformationstructures in a silk matrix or silk body, binding affinity of thetherapeutic agent to a silk matrix or silk body, and any combinationsthereof.

In addition, silk matrix or silk body can stabilize the bioactivity of atherapeutic agent under a certain condition, e.g., under an in vivophysiological condition. See, e.g., U.S. Provisional Application No.61/477,737, the content of which is incorporated herein by reference,for additional details on compositions and methods of stabilization ofactive agents. Accordingly, in some embodiments, encapsulating atherapeutic agent in a silk matrix or silk body can increase the in vivohalf-life of the therapeutic agent. For example, in vivo half-life of atherapeutic agent dispersed or encapsulated in a silk matrix or silkbody can be increased by at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 90%, at leastabout 1-fold, at least about 1.5-folds relative to the therapeutic agentwithout the silk matrix or silk body. Without wishing to be bound bytheory, an increase in in vivo half-life of a therapeutic agentdispersed or encapsulated in a silk matrix or silk body can provide alonger therapeutic effect. Stated another way, an increase in in vivohalf-life of a therapeutic agent dispersed or encapsulated in a silkmatrix or silk body can allow loading of a smaller amount of thetherapeutic agent for the same duration of therapeutic effect.

In some embodiments, at least one therapeutic agent can be dispersed orencapsulated in the silk matrix or silk body. In some embodiments, atleast two or more therapeutic agents can be dispersed or encapsulated inthe silk matrix or silk body. The therapeutic agent can be in any formsuitable for a particular method to be used for encapsulation and/ordispersion. For example, the therapeutic agent can be in the form of asolid, liquid, or gel. In some embodiments, the therapeutic agent can bein the form of a powder or a pellet. In some embodiments, thetherapeutic agent can be dispersed or encapsulated in a silk solutionbefore forming the silk matrix or silk body. In some embodiments, thetherapeutic agent can be dispersed or encapsulated in a silk solutionafter forming the silk matrix or silk body. For example, the therapeuticagent can be dispersed homogeneously or heterogeneously within the silkmatrix or silk body, or dispersed in a gradient, e.g., using thecarbodiimide-mediated modification method described in the U.S. PatentApplication No. US 2007/0212730. In some embodiments, the therapeuticagent can be coated on a surface of the silk matrix or silk body, e.g.,via diazonium coupling reaction (see, e.g., U.S. Patent Application No.US 2009/0232963), and/or avidin-biotin interaction (see, e.g.,International Application No.: WO 2011/011347). In some embodiments, thetherapeutic agent can be encapsulated in the silk matrix or silk body,e.g., by blending the therapeutic agent into a silk solution beforeprocessing into a desired material state, e.g., a silk tube. In someembodiments, the therapeutic agent can be present in a form of a fusionprotein with silk protein, e.g., by genetically engineering silk togenerate a fusion protein comprising the therapeutic agent.

In some embodiments, the therapeutic agent can be dispersed orencapsulated in a silk matrix or silk body after the silk matrix or silkbody is formed, e.g., by placing the formed silk matrix or silk body ina therapeutic agent solution and allowing the therapeutic agent diffuseinto the silk matrix or silk body over a period of time. In someembodiments, the silk matrix or silk body can be optionally hydratedbefore loading with the therapeutic agent. For example, the silk matrixor silk body can be incubated in deionized water until completelyhydrated.

Embodiments of the various aspects described herein can be illustratedby the following numbered paragraphs.

-   1. A silk-based implantable system comprising an electrical    component, wherein at least a portion of the electrical component is    in contact with a silk matrix, the silk matrix providing the    electrical component with sufficient stiffness to penetrate a target    tissue and becoming compliant upon the penetration.-   2. The silk-based implantable system of paragraph 1, wherein the    electrical component is at least part of a pre-formed implantable    device, wherein the pre-formed implantable device without the silk    matrix deforms during the penetration.-   3. The silk-based implantable system of paragraph 2, wherein the    pre-formed implantable device is a neuroprosthetic device.-   4. The silk-based implantable system of paragraph 3, wherein the    neuroprosthetic device includes a brain penetrating electrode, a    shunt, or a nerve guide.-   5. The silk-based implantable system of paragraph 1, wherein the    electrical component is patterned on the silk matrix.-   6. The silk-based implantable system of any of paragraphs 1-5,    wherein the electrical component includes a silk-based electrode.-   7. The silk-based implantable system of any of paragraphs 1-6,    wherein the silk matrix becomes compliant upon the penetration by    hydration of the silk matrix.-   8. The silk-based implantable system of paragraph 7, wherein the    silk matrix becomes compliant upon the penetration to provide    conformal contact between the electrical component and a surface of    the target tissue.-   9. The silk-based implantable system of any of paragraphs 1-8,    wherein at least one side of the electrical component is coated with    the silk matrix.-   10. The silk-based implantable system of any of paragraphs 1-9,    wherein the silk matrix comprises an active agent.-   11. The silk-based implantable system of paragraph 10, wherein the    active agent promotes tissue growth, controls inflammation at a site    of the penetration, or both.-   12. The silk-based implantable system of any of paragraphs 10-11,    wherein the active agent includes a gliosis-modulating agent.-   13. The silk-based implantable system of any of paragraphs 1-12,    wherein the silk matrix has a thickness of about 1 μm to about 1000    μm.-   14. The silk-based implantable system of any of paragraphs 1-13,    wherein the silk matrix increases a buckling force of the electrical    component or the implantable device by at least about 2-fold, as    compared to the absence of the silk matrix.-   15. The silk-based implantable system of any of paragraphs 1-14,    wherein the silk matrix reduces gliosis around the electrical    component or the implantable device by at least about 10%, as    compared to the absence of the silk matrix.-   16. A method of inserting a flexible or soft implantable device into    a target tissue comprising providing a silk-based implantable system    of any of paragraph 1-15.-   17. A method of reducing gliosis around a neuroprosthetic device    implanted in a brain tissue comprising providing a silk-based    implantable system of any of paragraphs 1-15.-   18. A method of improving long-term functionality of a    neuroprosthetic device implanted in a brain tissue comprising    providing a silk-based implantable system of any of paragraphs 1-15.-   19. The method of any of paragraphs 16-18, wherein the silk matrix    is in a dry state before implantation.-   20. A silk-based implantable device comprising a silk body with at    least one electrically-conducting component.-   21. The device of paragraph 20, wherein the silk body is a silk tube    with said at least one electrically-conducting component formed on    at least a portion of a lateral surface of the silk tube.-   22. The device of paragraph 21, wherein the silk tube is filled with    silk solution.-   23. The device of paragraph 20, wherein the silk body comprises a    first silk layer and a second silk layer, at least one of the first    and the second silk layer comprising at least one    electrically-conducting component formed on at least a portion of a    surface of the silk layer.-   24. The device of paragraph 23, wherein between the first and the    second silk layer includes silk particles or powders.-   25. The device of paragraph 24, wherein the silk particles or    powders are lyophilized.-   26. The device of paragraph 25, wherein the silk particles or    powders become a silk solution upon contact with a fluid.-   27. The device of any of paragraphs 20-26, wherein the    electrically-conducting component includes one or more through holes    such that the silk solution present in the silk tube or between the    silk layers is able to be discharged onto a surface of the    electrically-conducting component.-   28. The device of paragraph 27, wherein the silk solution on the    surface of the electrically-conducting component forms a gel upon    application of a first voltage through the electrically-conducting    component, and turns to a solution upon application of a second    voltage with a polarity opposite to the first voltage.-   29. The device of any of paragraphs 20-28, wherein the electrical    conducting component includes a metal.-   30. The device of paragraph 29, wherein the metal is a transition    metal or a noble metal.-   31. The device of paragraph 30, wherein the transition metal    includes silicon.-   32. The device of paragraph 30, wherein the noble metal is selected    from a group consisting of gold, platinum, titanium, and any    combinations thereof.-   33. The device of any of paragraphs 20-32, wherein the electrical    conducting component includes silk modified to conduct electricity.-   34. The device of paragraph 33, wherein the silk is functionalized    by modifying a tyrosine of the silk protein to a sulfate group    followed by polymerization of the modified tyrosine with a    conducting polymer.-   35. The device of paragraph 33 or 34, wherein the silk is doped with    a conductive material.-   36. The device of paragraph 35, wherein the conductive material is    selected from the group consisting of gold nanoparticles, carbon    nanotubes, graphene, and a conducting polymer.-   37. The device of paragraph 36, wherein the conducting polymer    includes polyethylenedioxythiophene (PEDOT), polypyrrole-based    conductive polymer, copolymers of thiophenes and polypyrroles,    copolymers of poly-lactide and polyaniline, or any combinations    thereof.-   38. The device of any of paragraphs 20-37, wherein the silk-based    implantable device is adapted for use as an implantable brain    penetrating electrode.-   39. The device of paragraph 38, wherein the electrode has a diameter    of less than 2 mm.-   40. The device of paragraph 38 or 39, wherein the electrode has a    tensile strength of at least about 2 MPa when the silk body is in a    dry state.-   41. The device of any of paragraphs 38-40, wherein the electrode has    a shear modulus of less than about 200 kPa upon contact of the silk    body with a fluid.-   42. The device of any of paragraphs 20-41, wherein the silk body    comprises an active agent.-   43. A method of regenerating a silk coating on a surface of a device    comprising providing the silk-based device of any of paragraphs    20-42, wherein the silk solution discharged onto the surface of the    electrically-conducting component forms a gel upon application of a    first voltage through the electrically-conducting component, and    turns to a solution upon application of a second voltage with a    polarity opposite to the first voltage.-   44. A method of reducing biofouling of a device comprising providing    the silk-based device of any of paragraphs 20-42, wherein the silk    solution discharged onto the surface of the electrically-conducting    component forms a gel upon application of a first voltage through    the electrically-conducting component, and turns to a solution upon    application of a second voltage with a polarity opposite to the    first voltage.-   45. The method of paragraph 43 or 44, wherein the device is placed    in vivo or in situ.-   46. The method of any of paragraphs 43-45, wherein the first voltage    and the second voltage is at least about 1.2V.-   47. The method of paragraph 46, wherein the first voltage and the    second voltage is about 5 V to about 50 V.

Some Selected Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±5% of the value being referred to. For example, about 100 meansfrom 95 to 105.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The term “flexible” or “soft” is generally used herein in reference to amechanical property of an implantable device. For example, in someembodiments, a flexible or soft implantable device can refer to animplantable device being not stiff enough to penetrate into a targettissue. In some embodiments, the term “flexible” or “soft” is used torefer to a mechanical state of an implantable device. For example, animplantable device becoming flexible or softer can refer to theimplantable device being compliant with a target tissue to bepenetrated.

As used herein, the terms “proteins” and “peptides” are usedinterchangeably herein to designate a series of amino acid residuesconnected to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and “peptide”,which are used interchangeably herein, refer to a polymer of proteinamino acids, including modified amino acids (e.g., phosphorylated,glycated, etc.) and amino acid analogs, regardless of its size orfunction. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Theterm “peptide” as used herein refers to peptides, polypeptides, proteinsand fragments of proteins, unless otherwise noted. The terms “protein”and “peptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary peptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

The term “nucleic acids” used herein refers to polynucleotides such asdeoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA), polymers thereof in either single- or double-stranded form.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides, which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer, et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608(1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)). Theterm “nucleic acid” should also be understood to include, asequivalents, derivatives, variants and analogs of either RNA or DNA madefrom nucleotide analogs, and, single (sense or antisense) anddouble-stranded polynucleotides.

The term “short interfering RNA” (siRNA), also referred to herein as“small interfering RNA” is defined as an agent which functions toinhibit expression of a target gene, e.g., by RNAi. An siRNA can bechemically synthesized, it can be produced by in vitro transcription, orit can be produced within a host cell. siRNA molecules can also begenerated by cleavage of double stranded RNA, where one strand isidentical to the message to be inactivated. The term “siRNA” refers tosmall inhibitory RNA duplexes that induce the RNA interference (RNAi)pathway. These molecules can vary in length (generally 18-30 base pairs)and contain varying degrees of complementarity to their target mRNA inthe antisense strand. Some, but not all, siRNA have unpaired overhangingbases on the 5′ or 3′ end of the sense 60 strand and/or the antisensestrand. The term “siRNA” includes duplexes of two separate strands, aswell as single strands that can form hairpin structures comprising aduplex region.

The term “shRNA” as used herein refers to short hairpin RNA whichfunctions as RNAi and/or siRNA species but differs in that shRNA speciesare double stranded hairpin-like structure for increased stability. Theterm “RNAi” as used herein refers to interfering RNA, or RNAinterference molecules are nucleic acid molecules or analogues thereoffor example RNA-based molecules that inhibit gene expression. RNAirefers to a means of selective post-transcriptional gene silencing. RNAican result in the destruction of specific mRNA, or prevents theprocessing or translation of RNA, such as mRNA.

The term “enzymes” as used here refers to a protein molecule thatcatalyzes chemical reactions of other substances without it beingdestroyed or substantially altered upon completion of the reactions. Theterm can include naturally occurring enzymes and bioengineered enzymesor mixtures thereof. Examples of enzyme families include kinases,dehydrogenases, oxidoreductases, GTPases, carboxyl transferases, acyltransferases, decarboxylases, transaminases, racemases, methyltransferases, formyl transferases, and a-ketodecarboxylases.

The term “vaccines” as used herein refers to any preparation of killedmicroorganisms, live attenuated organisms, subunit antigens, toxoidantigens, conjugate antigens or other type of antigenic molecule thatwhen introduced into a subjects body produces immunity to a specificdisease by causing the activation of the immune system, antibodyformation, and/or creating of a T-cell and/or B-cell response. Generallyvaccines against microorganisms are directed toward at least part of avirus, bacteria, parasite, mycoplasma, or other infectious agent.

As used herein, the term “aptamers” means a single-stranded, partiallysingle-stranded, partially double-stranded or double-stranded nucleotidesequence capable of specifically recognizing a selectednon-oligonucleotide molecule or group of molecules. In some embodiments,the aptamer recognizes the non-oligonucleotide molecule or group ofmolecules by a mechanism other than Watson-Crick base pairing or triplexformation. Aptamers can include, without limitation, defined sequencesegments and sequences comprising nucleotides, ribonucleotides,deoxyribonucleotides, nucleotide analogs, modified nucleotides andnucleotides comprising backbone modifications, branchpoints andnonnucleotide residues, groups or bridges. Methods for selectingaptamers for binding to a molecule are widely known in the art andeasily accessible to one of ordinary skill in the art.

As used herein, the term “antibody” or “antibodies” refers to an intactimmunoglobulin or to a monoclonal or polyclonal antigen-binding fragmentwith the Fc (crystallizable fragment) region or FcRn binding fragment ofthe Fc region. The term “antibodies” also includes “antibody-likemolecules”, such as portions of the antibodies, e.g., antigen-bindingfragments. Antigen-binding fragments can be produced by recombinant DNAtechniques or by enzymatic or chemical cleavage of intact antibodies.“Antigen-binding fragments” include, inter alia, Fab, Fab′, F(ab′)2, Fv,dAb, and complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single domain antibodies, chimericantibodies, diabodies, and polypeptides that contain at least a portionof an immunoglobulin that is sufficient to confer specific antigenbinding to the polypeptide. Linear antibodies are also included for thepurposes described herein. The terms Fab, Fc, pFc′, F(ab′) 2 and Fv areemployed with standard immunological meanings (Klein, Immunology (JohnWiley, New York, N.Y., 1982); Clark, W. R. (1986) The ExperimentalFoundations of Modern Immunology (Wiley & Sons, Inc., New York); andRoitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell ScientificPublications, Oxford)). Antibodies or antigen-binding fragments specificfor various antigens are available commercially from vendors such as R&DSystems, BD Biosciences, e-Biosciences and Miltenyi, or can be raisedagainst these cell-surface markers by methods known to those skilled inthe art.

As used herein, the term “Complementarity Determining Regions” (CDRs;i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region maycomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e. about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e. about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop.

The expression “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

The expression “single-chain Fv” or “scFv” antibody fragments, as usedherein, is intended to mean antibody fragments that comprise the VH andVL domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. (The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994)).

The term “diabodies,” as used herein, refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy-chainvariable domain (VH) Connected to a light-chain variable domain (VL) inthe same polypeptide chain (VH-VL). By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. (EP 404,097; WO 93/11161; Hollinger etah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).

The term “antibiotics” is used herein to describe a compound orcomposition which decreases the viability of a microorganism, or whichinhibits the growth or reproduction of a microorganism. As used in thisdisclosure, an antibiotic is further intended to include anantimicrobial, bacteriostatic, or bactericidal agent. Exemplaryantibiotics include, but are not limited to, penicillins,cephalosporins, penems, carbapenems, monobactams, aminoglycosides,sulfonamides, macrolides, tetracyclines, lincosides, quinolones,chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid,spectinomycin, trimethoprim, sulfamethoxazole, and the like.

As used herein, the term “antigens” refers to a molecule or a portion ofa molecule capable of being bound by a selective binding agent, such asan antibody, and additionally capable of being used in an animal toelicit the production of antibodies capable of binding to an epitope ofthat antigen. An antigen may have one or more epitopes. The term“antigen” can also refer to a molecule capable of being bound by anantibody or a T cell receptor (TCR) if presented by MHC molecules. Theterm “antigen”, as used herein, also encompasses T-cell epitopes. Anantigen is additionally capable of being recognized by the immune systemand/or being capable of inducing a humoral immune response and/orcellular immune response leading to the activation of B- and/orT-lymphocytes. This may, however, require that, at least in certaincases, the antigen contains or is linked to a Th cell epitope and isgiven in adjuvant. An antigen can have one or more epitopes (B- andT-epitopes). The specific reaction referred to above is meant toindicate that the antigen will preferably react, typically in a highlyselective manner, with its corresponding antibody or TCR and not withthe multitude of other antibodies or TCRs which may be evoked by otherantigens. Antigens as used herein may also be mixtures of severalindividual antigens.

The term “immunogen” refers to any substance, e.g., vaccines, capable ofeliciting an immune response in an organism. An “immunogen” is capableof inducing an immunological response against itself on administrationto a subject. The term “immunological” as used herein with respect to animmunological response, refers to the development of a humoral (antibodymediated) and/or a cellular (mediated by antigen-specific T cells ortheir secretion products) response directed against an immunogen in arecipient subject. Such a response can be an active response induced byadministration of an immunogen or immunogenic peptide to a subject or apassive response induced by administration of antibody or primed T-cellsthat are directed towards the immunogen. A cellular immune response iselicited by the presentation of polypeptide epitopes in association withClass I or Class II MHC molecules to activate antigen-specific CD4+Thelper cells and/or CD8+ cytotoxic T cells. Such a response can alsoinvolve activation of monocytes, macrophages, NK cells, basophils,dendritic cells, astrocytes, microglia cells, eosinophils or othercomponents of innate immunity.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviation (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

As used interchangeably herein, the terms “essentially” and“substantially” means a proportion of at least about 60%, or preferablyat least about 70% or at least about 80%, or at least about 90%, atleast about 95%, at least about 97% or at least about 99% or more, orany integer between 70% and 100%. In some embodiments, the term“essentially” means a proportion of at least about 90%, at least about95%, at least about 98%, at least about 99% or more, or any integerbetween 90% and 100%. In some embodiments, the term “essentially” caninclude 100%.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedmay be further modified to incorporate features shown in any of theother embodiments disclosed herein.

The disclosure is further illustrated by the following examples whichshould not be construed as limiting. The examples are illustrative only,and are not intended to limit, in any manner, any of the aspectsdescribed herein. The following examples do not in any way limit theinvention.

EXAMPLES Example 1: Exemplary Materials and Methods

Preparation of Silk Solution.

Silk solution was prepared from Bombyx mori silkworm cocoons accordingto the procedures described in previously-reported studies (35-37).Cocoons of B. mori silkworm silk were supplied by Tajima Shoji Co.(Yokohama, Japan). Briefly, the cocoons were degummed in a boiling0.020□M Na2CO3 (Sigma-Aldrich, St. Louis, Mo.) solution for 15 min. Thefibroin extract was then rinsed three times in Milli-Q water, dissolvedin a 9.3M LiBr solution yielding a 20% w/v solution, and subsequentlydialyzed (MWCO 3,500 kDa) against distilled water for 2 days to obtainregenerated aqueous silk fibroin solution (ca. 8% w/v). The silksolution was concentrated to 15% w/v by dialysis overnight.

Fabrication of Electrodes.

Flexible electrodes (e.g., flexible cortical electrodes) could befabricated by any standard MEMS fabrication techniques known in the art.In some embodiments, any art-recognized flexible electrodes can be usedin the systems, devices and/or methods described herein. In someembodiments, any commercially-available flexible electrodes can be used.

Coating of Electrodes.

Flat polydimethylsiloxane (PDMS) molds were prepared using two differenttechniques. For a rapid prototyping approach, 3-5 mm of Sylgard 184 (DowCorning Corp., Midland, Mich.) was cast on petri dishes. Upon curingovernight at 60° C., the PDMS was cut into molds of varying design usinga commercial laser cutter. PDMS molds were also prepared usingconventional soft lithography microfabrication techniques (45), whichallowed for finer control of edge morphology and features. A dissectingmicroscope was used to manually align the electrodes on the PDMS molds.The electrodes were coated by dipping the head of a pin into 15% w/vaqueous silk solution and drawing the bead of silk down the mold andelectrode shank from the base to the tip, as a modification of ourlayer-by-layer technique (Wang et al., 2005). Coatings were allowed todry for 15 minutes before applying another layer.

Microscopy.

Coated probes were imaged using the 10× objective on a Zeiss Axiovert 40CFL (Carl Zeiss AG, Germany) microscope. Dimensions for both 3 and 6layer coatings of silk fibroin were measured with QCapture Pro software.Coated probes could be also imaged using scanning electron microscopy.

Buckling Force Test.

Coated and uncoated probes were tested in ambient conditions todetermine their buckling force (the maximum force exerted by the probeswhen pinned and loaded in a vertical orientation). This characterizationwas chosen to mimic the type of loading experienced by the electrodesduring insertion into the brain. A custom clamping mechanism wasfabricated to fix the probes to the crosshead of an Instron 3366mechanical testing frame. Samples were lowered at a rate of 0.5 mm/minonto the weigh plate of a Mettler-Toledo MS204S/03 analytical balance.The balance was capable of reading mass to the 10th of a milligram,allowing milli-Newton forces to be recorded with custom software thatinterfaced with the scale.

Euler's fixed-pin beam buckling equation (10,12):

$F_{b} = \frac{\Pi^{2}E_{silk}\frac{1}{12}\left( {bh}^{3} \right)}{\left( {0.7L} \right)^{2}}$

was utilized to fit a curve to the measured buckling force and probethicknesses using nonlinear regression with the elastic modulus as theindependent variable, where F_(b) is the buckling force, E_(silk) is theelastic modulus, h is the thickness of the probe, b is the width (400μm) and L is the length (5.5 mm).

Brain Phantom Insertion Test.

Insertion tests were performed using the Instron crosshead and customclamp to lower coated and uncoated probes at a speed of 4 mm/min into0.6% agar, a close match to the mechanical properties of the brain (46).The outcome following insertion was photographed.

In Vitro Glial Scar Model Cell Culture.

Glial scarring around silk-coated microwires was assessed using a 2Dmodel based on the protocol developed by Polikov et al. (47, 48, 54).Briefly, the cortices of embryonic day 18 rat pups were dissociated in0.5% trypsin and plated in 24 well, poly-L-lysine coated plates at adensity of 0.5 million cells/well. The cells were incubated at 37° C. inNeurobasal medium containing B27 supplement, 1% pen-strep, 1% GlutaMax(Invitrogen, Inc., Carlsbad, Calif.), and 10 ng/mL beta fibroblastgrowth factor (bFGF, R&D Systems, Minneapolis, Minn.). 10 days afterseeding, the media was changed to include 10% FBS (Invitrogen) and 2-3segments of steel microwire, prepared as detailed below, were allowed tosink to the bottom of each well and rest on the dissociated brain cellcultures.

Cultures with wires were fixed 17 days after seeding by incubating in 4%paraformaldehyde for 30 minutes. After washing with PBS, cells werepermeablized and blocked with PBS containing 4% goat serum (Invitrogen)and 0.1% Triton-X. Primary antibodies for glial fibrillary acid protein(GFAP) (rabbit anti-GFAP, Sigma) and chondroitin sulfate (CS) (mouseanti-CS, Sigma) were diluted 1:1000 in permeablization buffer andincubated with the cells overnight at 4° C. After three PBS washes,cells were incubated with horseradish peroxidase conjugated secondaryIgG antibodies specific for rabbit and mouse, respectively (Santa CruzBiotechnology, Santa Cruz, Calif.), diluted 1:500 in permeablizationbuffer for 2 hours. Color was developed using a DAB substrate kit(Sigma), according to the manufacturer's instructions and incubated withcells for 1 hour.

Steel Microwire Coating.

Stainless steel microwire with a diameter of 50 μm (A-M Systems, Sequim,Wash.) was used for the scarring assay. The four treatment groups forthe wire consisted of uncoated wire, silk coated wire, uncoated wiredipped in cytarabine (Ara-C) (Sigma), and silk coated wire with Ara-C.Silk coatings were achieved by dipping the microwire into ˜7% w/v silksolution, allowing the silk to air dry on the wire for 15 minutes, andwater annealing for 20 minutes to crystallize the silk. In oneembodiment, the dipping procedure was repeated 5 times in order to buildup a 5-10 micrometer thick coating of silk around the circumference ofthe wire. Uncoated wires were treated in the same manner, but dipped inmilli-Q water instead of silk solution. For the drug-loaded wires, Ara-Cwas dissolved into the silk solution or milli-Q water at a concentrationof 25 mg/mL prior to dipping. The microwire was cut into 3-4 mm segmentsprior to adding to cultures as specified above.

Glial Scar Model Analysis and Quantification.

After fixing and staining cells, images were taken along the entirelength of each microwire using a 10× objective on a Zeiss Axiovert 40CFL (Carl Zeiss AG) microscope. The images were analyzed in an automatedfashion using custom ImageJ (NIH, Bethesda, Md.) and Matlab (Mathworks,Natick, Mass.) scripts based on the methods of Polikov et al. (48).Briefly, pixel intensity was averaged across the entire width of eachimage with the microwire registered on the horizontal. The position ofthe microwire was determined to be the darkest 50 μm portion of theimage. The background pixel intensity was quantified by averaging the100 μm of pixels farthest from the wire at each edge of the image, andthen averaging the values for each side of the wire. The scar index foreach wire was defined as the area enclosed between the edge of themicrowire and the averaged pixel intensity curve at the point where theintensity reached 90% of the previously determined background value. Inthis way, the scar index provides a quantitative means to assess theintensity of staining proximal to the microwires.

In Vitro Screening for Cell Response to an Implantable System/Device:

Astrocytes and PC12 cells can be used to screen the implantabledevices/systems described herein in vitro under a controlled environmentto study responses of the cells to the implantable devices/systemsdescribed herein (e.g., electrodes). In addition, the implantabledevices/systems described herein can be evaluated for their ability torecord signals in vitro.

Temperature modulated differential scanning calorimetry (TMDSC):

An advanced thermal analysis method can be used to determine the glasstransition, crystallization, and thermal degradation properties of silksamples.

Fourier Transform Infrared spectroscopy (FTIR):

FTIR can be selected to analyze the silk structures prepared underdifferent conditions. An art-recognized protein structural predictiontechnique, based on Fourier Self-Deconvolution (FSD) of the infraredspectra covering the Amide I region (1595-1705 cm⁻¹), can be used tocalculate the fraction of the secondary structure.

Characterization of Mechanical Properties:

Samples can be tested for mechanical properties, including tensiletesting, stress strain characterization, thermo-mechanical properties,cross-linking density, and/or the swelling ratio. Routinemacro-mechanical assessments of the materials can include traditionalInstron compression tests and an Instron Video Extensometer can be usedand initial elastic modulus, yield stress, tensile strength, andelongation ratio can be determined.

Scanning Electron Microscopy (SEM):

SEM can be used to examine the silk-based device in variable stages ofpreparation and degradation to assess morphologies. The samples can befixed for 24 h in 0.4% glutaraldehyde after fractured in liquid nitrogenusing a razor blade and then dehydrated in a series of graded ethanolextractions prior to coating with gold/palladium for 3 min before SEMobservation.

Statistical Analysis.

Data is presented in graphs as average ±standard deviation. Averagevalues were obtained from 3 separate replicates, unless noted otherwise.Data was analyzed for significance (p<0.05) with GraphPad Prism software(GraphPad Software, Inc., La Jolla, Calif.) using a two-tailed unpairedt-test.

Example 2. Exemplary Coating of Flexible Neural Probes with Silk Fibroin

A layer-by-layer casting technique was used to coat a flexibleelectrode, e.g., a polyimide-based thin film neural probe) with silkfibroin. The probes were first centered, with electrode recording sitesfacing down, on PDMS molds that were fabricated using a laser cutter orsoft lithography techniques. The shape of the PDMS mold can vary withthe shape and/size of the electrode. The PDMS mold can partly determinethe silk-coated electrode shank dimensions. Concentrated silk solution(15% w/v) was applied to the molds, e.g., by drawing a bead of solutionfrom the tip of a pin head along the length of the probe shank. The drawspeed and viscosity of the silk solution, as well as the mold shape,determined the amount of solution that adhered to the mold and probe.After drying, successive layers of silk were applied in the same mannerin order to increase the thickness of the coating. Despite the manualnature of this coating technique, results were relatively consistent.For blunt tipped, 400 μm wide molds, 3 layers of silk produced a finalthickness of −70 μm on average and 6 layers resulted in twice thethickness, −140 μm on average. In some embodiments, one single side ofthe electrode can be coated with this method, thus allowing recordingsites to remain exposed. The silk electrode shank thickness can becontrolled partly by the number of coatings. Without wishing to bebound, the layer-by-layer coating method can be performed manually orautomated by a machine.

In some embodiments, silk coatings were applied to pre-fabricatedthin-film electrodes via layer by layer casting. The coating isconformal and the thickness is controllable based on the concentrationof the silk solution and the number of layers applied. Because thecasting steps are carried out using PDMS molds, the width and shape ofthe coating can be adjusted using various well-defined rapid prototypingand microfabrication techniques to pattern the underlying PDMS. Thecoating technique described herein can be amenable to a variety ofgeometries. It is reported that the tip shape and surface andcross-sectional area of penetrating probes can affect the glial response(49, 50). Additionally, in contrast to dipping based coating methods,the layer-by-layer casting technique can produce a unilateral coating,allowing the recording sites on the underside of the probe (e.g., onlyone side of the probe shank) to remain partially or completely exposed.This can increase the recording capabilities of the probe afterimplantation, and reducing or eliminating concerns for the silkelectrically or physically insulating the electrodes from neurons whenimplanted in vivo.

In some embodiments, aside from the modes of coating utilized in thisExample, there can include options for post-coating treatments, whereinthe mechanical features of the silk can be further modulated to impactthe initial properties as well as the rate of hydration upon insertion.Exemplary post-coating treatments can include additional pre-dryingsteps (e.g., dry nitrogen), exposure to methanol (e.g., to maximizecrystallinity) and other treatment such as electric fields andmechanical shear to induce different outcomes.

Example 3. Mechanical Properties of Silk-Coated Probes

Uncoated, 3-layer, and 6-layer silk-coated probes can be mechanicallycharacterized by driving the probes perpendicularly onto a precisionanalytical balance at a rate of about 0.5 mm/min. Without wishing to bebound by theory, there can be an initial linear increase in force whilethe probes remain straight before buckling. As the probes continue to bedriven onto the balance, the shanks can begin to buckle and bend. Thiscan be characterized by the peak and gradual decrease in force.Eventually, the silk coating on the 3 and 6 layer probes can fracturemidway down the shank, which can be characterized by a sharp decrease inforce exerted on the balance.

For practical in vivo applications, neural probes are desired to remainstraight during insertion in order to reach a precisely targeted regionof the brain. Therefore, the buckling force should not be exceededduring insertion through the pia mater and into the cortex. The averagebuckling force was approximately 42 μN for the uncoated probe, ˜12 mNfor the 3 layer silk-coated probe, and 1˜05 mN for the 6 layer coatedprobe. The difference in buckling forces can be at least partly resultedfrom the different mechanical properties of the silk coating, as well asthe different dimensions of the probes imparted by the mold and layeringof silk.

When approximated as rectangular beams, the buckling mechanics of thesilk-coated electrodes can be modeled using Euler's fixed pin bucklingequation (see Example 1). Using non-linear regression with the electrodeelastic modulus as the independent variable, Euler's equation can be fitto a plot of the thickness and buckling force of the 3 and 6 coatedelectrodes, and determine the elastic modulus of silk (E_(silk)), e.g.,approximately 1.5 GPa under ambient conditions, when the silkcontributes the majority of the mechanical properties to the silk-coatedelectrodes.

In the dry state, the layered silk coatings allow the probes to sustainmuch greater forces before buckling, relative to the uncoated state.Without wishing to be bound by theory, the buckling force isproportional to the cube of the device thickness, thus a small change inthickness can produce a large change in the buckling force. For example,the 6-layer coated probes buckle at a force a full order of magnitudehigher than the 3-layer coated probes, despite being only twice asthick. The E_(silk) of ˜1.5 GPa predicted by the fit of Euler's beambuckling equation to the experimental data is on the same order ofmagnitude as previous reports of the modulus of dry silk films (51),providing validation for the use of this model to predict the bucklingforce for various silk coated probe dimensions. While features such astapered tip geometries can affect the buckling mechanics, the Euler'sbeam buckling model can provide a good point of reference for designingprobe coatings with the necessary dimensions to penetrate the pia ordura mater.

Example 4. In Vitro Brain Phantom Insertions

Uncoated, 3 layer, and 6 layer silk coated probes were inserted into anagar gel mechanical brain phantom in order to test the efficacy of thesilk coating for facilitating electrode insertion. The electrodes wereclamped to the Instron crosshead in the same manner as for the bucklingforce test and lowered onto the gel surface at a rate of 4 mm/min. Theuncoated probe was unable to penetrate the surface of the gel. The 3layer silk-coated probe penetrated the gel surface, but bent and curledwithin the gel as the insertion continued. Finally, the 6 layersilk-coated probe penetrated the gel and remained straight during theentire insertion.

Previous reports have shown the elastic modulus of hydrated silk filmsto be on the order of 20 MPa (52), a decrease of nearly two orders ofmagnitude compared to the dry state, and similar to the mechanicalproperties demonstrated for other proposed electrode materials (12).Without wishing to be bound by theory, such hydration-mediatedtransition in mechanics can reduce the strain concentration at thesilk-coated electrode-tissue interface, improving long-term reliability.This example also shows the mechanically dynamic nature of the probes.While the 3-layer coated probe was stiff enough to penetrate the gelinitially, during the course of insertion the tip of the probe hydratedbefore the shaft was fully inserted, causing bending within the gel. Thethicker, 6 layer coated probe took longer to hydrate and transition to amore flexible state, thus allowing it to fully penetrate into the gelwhile remaining straight. Based on these findings, relatively rapidinsertion speeds can be desirable to allow the electrode shank to fullypenetrate the brain before becoming too flexible due to hydration.Further, previous reports have shown that faster insertion speeds maycause less acute damage to the brain (53).

Example 5. Assessing Silk-Mediated Gliosis with an In Vitro Model

The effect of silk fibroin coatings on reactive gliosis was tested in anin vitro model of glial scarring around an electrode mimic. Uncoated andsilk-coated segments of 50 μm stainless steel microwire electrodes weredropped into mixed cultures of dissociated embryonic rat cortical cells.Serum and bFGF were added to the culture to initiate the scarringresponse around the microwires and after one week the cultures werefixed and stained for glial fibrillary acid protein (GFAP) (FIGS. 1A and1B) and chondroitin sulfate (CS) (FIGS. 1C and 1D).

The scarring around the uncoated and coated wires was quantified basedon staining intensity proximal to the wires (FIGS. 2A and 2B). For GFAP,no difference in scar formation was observed between the uncoated andsilk-coated wires (FIG. 2A). However, the silk-coated wires stimulatedsignificantly less CS accumulation during scar formation relative to theuncoated wires (FIG. 2B).

In addition to the dynamic mechanical properties, the cellular responseto silk coatings in the in vitro model of glial scarring furthersupports its use in penetrating electrodes. Silk coatings resulted inthe same average amount of GFAP expression, a common marker for gliosisand astrocyte reactivity, relative to the uncoated inert stainless steelmicrowires. This indicates that astrocyte proliferation and activationwas similar for both materials. However, silk coatings stimulatedsignificantly less CS accumulation around the microwire. This surprisingfinding indicates that secretion of ECM molecules (e.g., inhibitory ECMmolecules such as CS) can be independent of astrocyte reactivity and canbe mediated by the material chemistry of the electrode implant. CS caninhibit axon growth and regeneration; therefore, lower levels of thismolecule can be beneficial for promoting neuronal proximity to electrodeimplants, improving long-term performance.

In addition, in some embodiments, nerve impulses can be measured byusing a silk-coated cortical electrode (e.g., for nerve recording) in abrain slice, and the scarring response can be determined as describedabove. A comparison of the silk-coated cortical electrode with existingcortical electrodes (e.g., without silk coating) can be conducted todetermine the performance and efficacy.

Example 6. Capacity for Silk to Release Gliosis Modulating Drugs InVitro

While silk coatings alone can reduce some gliosis responses compared tostainless steel, the ability to encapsulate and release scar-inhibitingcompounds can provide an additional avenue for improving the chronicreliability of silk-based penetrating electrodes. Accordingly, thescarring assay as shown in Example 5 was also carried out with theaddition of the anti-mitotic drug cytarabine (Ara-C) in the silkcoating. The drug was incorporated into the silk-coated microwires bydissolving it in the aqueous silk solution before dipping. Uncoatedcontrol wires were dipped in water loaded with the drug at the sameconcentration as in the silk solution. The silk-coated, Ara-C loadedwires produced a significantly different scarring response both in GFAP(FIG. 1F) and CS (FIG. 1H) expression relative to both silk alone andthe uncoated wire (FIGS. 1E and 1G).

CS quantification shows that Ara-C loaded silk coatings resulted in asignificantly lower scarring response relative to any other treatmentgroup, including silk alone and the Ara-C water dipped steel wires (FIG.2B).

The biocompatibility of chronic brain penetrating electrodes is integralto the long-term reliability and functionality of the device. Themechanical properties, innate immunogenicity, and/or chemicalfunctionalization, of the materials used to fabricate the probe shankscan affect the tissue response in vivo.

As presented herein, silk possesses a unique combination of materialproperties which make it suitable for use in a penetrating electrodesystem. The mechanical and low inflammatory properties of silk, as wellas its capacity to encapsulate and release gliosis-modifying compounds,make it an attractive biomaterial for the next generation of chronic,indwelling neural probes. Such probes can have improvedbiocompatibility, for reduced glial scar encapsulation and ultimatelybetter long-term reliability. The silk coating can have applicationsbeyond penetrating electrodes, e.g., the use of silk fibroin for manyother CNS applications, such as in shunts and spinal cord nerve guides,where gliosis is desirable to be controlled at the tissue-implantinterface to achieve optimal outcomes.

Further, provided herein is a novel dynamic biomaterial system forneural implant devices as a route to overcome loss of function at thebiological abiotic interface. Electrogelation of a silk solution can beused to generate conformal contact with nerves, to remodel the interfaceon demand, and to maintain electrical contact between the biomaterialand the biological tissue to optimize function over time.

Example 7. In Vivo Evaluation of an Implantable Device Described Herein

In some embodiments, nerve impulses can be measured in a brain tissue ofan animal model, e.g., a rat, by inserting a silk-coated corticalelectrode (e.g., for nerve recording) in its brain, and the scarringresponse can be determined as described above, e.g., by histology aftersacrifice. A comparison of the silk-coated cortical electrode withexisting cortical electrodes (e.g., without silk coating) can beconducted to determine the performance and efficacy.

Example 8. Use of Silk Coating to Strengthen a Flexible Wire with aTransition from Stiff to Compliant Upon Hydration

Flexible enameled copper wires (˜100 μm diameter) were dip-coated inhexafluoroisopropanol (HFIP) containing ˜15% w/v silk fibroin. Uponevaporation of the HFIP solvent, the wires were encapsulated in a stiffsilk coating (see FIG. 4). In order to demonstrate the dynamicmechanical properties of the coating, a beaker was filled with water andcovered with Parafilm to simulate a tissue membrane (such as the dura inthe brain). FIG. 5A shows an uncoated wire incapable of penetrating theParafilm. FIG. 5B shows that the silk-coated wire could easily penetratethe Parafilm membrane. The silk-coated wire after penetration was thenleft to hydrate in the aqueous environment for approximately 30 seconds.As shown in FIG. 5C, when the coated wire was removed from the water,the coated wire had transitioned to a flexible state and thus could notpenetrate the Parafilm membrane, as it was in a dry state before.

Example 9. Assessment of Various Coating Techniques to Generate SilkCoatings on Flexible Planar Substrates

Numerous techniques were assessed to determine suitable or optimumapproach for coating flexible implants. Strips of parylene C(approximately 2 mm×8 mm×60 μm), an inert material commonly used inmedical devices and in the fabrication of thin, flexible electrodes, wasused as a model test substrate. Coating methods generally is dividedinto two categories: (i) dip coating, and (ii) casting. The dip coatingapproaches involved briefly immersing the parylene strips in variousforms of silk solution, followed by air-drying (see FIG. 6A). Thesolutions utilized for dip coating included (i) aqueous silk solution,(ii) high silk concentration HFIP solution, and (iii) heat-liquefiedsilk electro-gel (e-gel) solution.

Alternatively, casting methods involved submerging the parylene stripsin a volume of aqueous or gel based silk solution spread on apolystyrene plate and allowing the silk to air dry to form films (seeFIGS. 6B-6C). The coated parylene was then manually cut out of theencapsulating film using a razor blade or scissors. For more preciserelease of the coated parylene from the silk film, a computer controlledlaser cutter could be employed.

In particular embodiments, the casting method can involve a two-steplayering process. First, a volume of heat-liquefied silk e-gel waspoured into a plate and allowed to re-gel by cooling (3-5 minutes). Theparylene strips to be encapsulated were then placed on top of the firste-gel layer and covered by another volume of heat-liquefied e-gel. Thelayered gel was then left to air dry. This process ensured even layersof silk on both sides of the parylene strips, preventing delamination ofthe silk from the substrate upon drying. The coating formed uniformlayers on the parylene, the thickness of which could be individuallycontrolled by adjusting the volume or concentration of the silk solutionapplied in each layer (see FIGS. 7A-7B and 8). Furthermore, theviscosity of the liquefied e-gel solution allowed for selective andlocalized application of the top layer. This enabled portions of theimplantable device to be left uncoated as desired. For instance, asshown in FIG. 9, only one side of the contact pads on an electrode wascoated with silk. In the future, cytokines could be incorporated intothe aqueous silk, which would become entrapped upon drying and releasedafter implantation.

Example 10. Buckling Force Characterization of Dynamic Silk Coatings

The mechanical properties of the uncoated and silk-coated implant models(e.g., parylene implants) were quantified by measuring the bucklingforce (the maximum force exerted by an implant, e.g., parylene strips,when pinned and loaded in a vertical orientation). This characterizationwas chosen to mimic the type of loading experienced by column-likeimplants, such as electrodes, when used to penetrate tissues. A customclamping mechanism was fabricated to fix a 5 mm length of the parylenestrips to the crosshead of an Instron 3366 mechanical testing frame.Samples were lowered at a rate of 0.5 mm/min onto a steel plate. Allmeasurements were performed in air at room temperature. Forcemeasurements were collected with an Instron 10 N load-cell and theaccompanying Bluehill Software package. When measured with low loads,e.g., for uncoated samples, force measurements of the uncoated sampleswere alternatively collected using a Mettler-Toledo MS204S/03 analyticalbalance and custom software. The values collected with this custom testconfiguration were shown in FIG. 10A after validating that they matchedclosely with the averaged values of the load-cell data.

Five samples from each of the three different treated implant groupswere evaluated (see FIGS. 10A-10C). The first group consisted ofuncoated parylene strips (approximately 2 mm×5 mm×60 μm) (FIG. 10A). Thehighly elastic strips quickly buckled at loads on the order oftwo-hundredths of a Newton. Parylene strips coated in silk(approximately 2.7 mm×5 mm×200 μm) were then evaluated in the dry state,and observed to buckle under loads on the order of 5 Newtons (FIG. 10B).Silk-coated samples were also evaluated after being allowed to hydratefor 30 minutes in 0.1M PBS at 37° C. (FIG. 10C). The samples wereimmediately mounted and tested after removal from the PBS, and buckledat forces on the order of 2-tenths of a Newton.

Average buckling forces for the three groups are shown on a logarithmicplot (FIG. 11). The dry silk-coated model implants withstood forces overtwo orders of magnitude larger than the uncoated samples before theybuckled. In an aqueous environment, the silk coating hydrated in amatter of minutes. This resulted in more than an order of magnitudesmaller buckling force compared to the dry silk-coated samples. Afterhydration for 30 minutes, the silk-coated samples have mechanicalproperties more similar to the uncoated samples, than to the coatedsamples in the dry state. Without wishing to be bound by theory, in someembodiments, because silk is fully biodegradable, longer periods in anaqueous environment can result in the properties of the coated samplesbecoming similar to those of the uncoated samples as the silk continuesto hydrate, and eventually degrades.

Example 11. Design of Implantable Silk Shanks and Silk-Coated Electrodes

Silk shanks can be produced by any methods known in the art. Forexample, a molding/freezing processing approach can be used to makerobust silk constructs. The molding/freezing approach involved theutilization of an aqueous silk solution made from cocoons from aTaiwanese supplier. The silk solution was concentrated to about 25% w/vfibroin (balance is water). Small-diameter, flexible Tygon tubing wasused as a mold; silk solution was injected using a plastic syringe andboth ends sealed using the high heat of a soldering iron. Thesilk-containing tubing was stored in an EdgeStar Model FP430thermoelectric cooler for a period of at least 1 week at −5° C. Uponremoval from the freezer, the molded material was released from thetube, e.g., by flushing the inner diameter with milli-Q water ejectedfrom a syringe. At the cooler temperature (−5° C.), the final samplemorphology can be determined by the length of time in the cooler. Forexample, after about 1.5 weeks in the cooler, the silk immediately afterremoval can be white, stretchable, e.g., with the general consistency ofboiled spaghetti. After storing at room temperature, trapped waterevaporates and the resulting material can become non-porous and fairlystiff. Varying diameters were achieved based on appropriate tubingselection (FIG. 13A). A sharp tip could be produced, e.g. by any knownmethods in the art, such as sharpening with a razor blade (FIG. 13B).

A different silk morphology could be achieved by leaving thesilk-containing tubing in the cooler at −5° C. for over 2 weeks (FIGS.13C-13D). Immediately after removal from the cooler, the silk wasflushed from the tubing and appeared to have a foam-like morphology. Itis known that the freezer temperature of water in silk solution is closeto −8.5° C. Without wishing to be bound by theory, the −5° C.temperature allows the water to start freezing, but allows the silkfibroin to maintain some mobility. Freeze-concentration occurs, in whichthe low surface-tension silk fibroin coagulates into specific regions ofhigh concentration and is stretched as freezing water begins to expand.The stretching and alignment of silk fibroin molecular chains leads tobonding and the formation of a porous silk network, from which the wateris ultimately removed. As FIG. 13C shows, the silk foam construct can bemolded with a conducting wire inside the tubing (top construct showssmall-diameter motor wire protruding from the left end of the sample).Thus, a tubular structure can be formed after removal of the conductingwire. Given this foam is fairly stiff when dry, a razor blade could beused to form a sharp tip (bottom construct in FIG. 13C). As with thesilk constructs described earlier, the diameter can be adjusted throughappropriate tubing selection (FIG. 13D).

To evaluate the ability of the sharpened silk constructs to penetratematerial, in one embodiment, Parafilm (a polymeric material used inlaboratories to cover and seal bottles and Petri dishes) was stretchedacross the open top of a plastic Petri dish. The sharpened nonporoussilk construct (without a wire embedded) or foam-like silk constructpenetrated the Parafilm membrane. No visible damage to the silk wasevident. However, all of the nonporous and foam-like silk constructsbecome softer when hydrated. Given its porous nature, the foam-likeconstructs became much softer than the nonporous constructs whenhydrated. When dried again, all constructs regained the stiffness asoriginally in a dry state.

Example 12. Electrogelation of a Silk Solution

When aqueous solutions of silkworm silk were exposed to DC currents,under certain electric fields, the solution begins to gel on thepositive electrode (FIGS. 14A-14B). While electrospinning of polymers,including silks, is generally performed at voltage potentials as highas >30 kV, the utilization of low DC voltages to generate a controlledvolume of silk gel was unexpected and novel. For an illustrative purposeonly, electrodes are immersed in an aqueous solution of silk protein and25 VDC is applied over a 3 minute period to a pair of leads.Electrolysis occurs during electrogelation. Within seconds of theapplication of the voltage, a visible gel forms at the positiveelectrode and emanates outward.

When silk electrogelation (e-gel) is executed in a voltage-controlledmanner, the current draw in the process follows a repeated trend;initially high current draw drops exponentially to a minimal level. Theactual current values depend on many factors, including applied voltagelevel, electrode area and spacing, and conductivity of the silksolution. Silk gel formed through electrogelation has a highly viscous(soft) consistency and is very tacky, bearing a resemblance to thickmucus. Remarkably, after the electric field was turned off, the adhesivegel state was retained. Thus, the structural state of the protein formedunder e-gel conditions was sufficiently stable to retain materialfunctions in the absence of the applied electric field. Yet, the gelformed can be returned to the solution state through a reverseelectrical process. If the electrode polarity is reversed and 25 VDC isre-applied, the gel disappears, while fresh gel is formed on the newlycreated positive electrode. Electrogelation and reversal back to silksolution can be cycled many times.

The changes in mechanical characteristics due to electrogelation of silksolutions were evaluated by dynamic oscillatory shear rheology (FIGS.15A-15D). For silk solutions, liquid-like, viscous behavior measured bythe loss modulus (G″) dominated the mechanical response within theprobed frequency range ω with G″˜ω. On the other hand, the mechanicalresponse of e-gels resembled that of a soft-solid-like, physical gel.There was a significant increase in the elastic response, measured bythe storage modulus (G′). The frequency dependence of G′ was weak butfinite (G′˜ω^(0.1)), while the apparent minimum in G″ indicated apossible G′, G″ cross-over at even lower frequencies due to eventualrelaxation of temporary, physical crosslinks.

To investigate the significance of increased proton concentration in themechanism of e-gel formation at the positive electrode, the solution wastitrated to control the pH, termed “pH-gels”. After titration of thesilk solution, pH-gels displayed viscoelastic behavior where G′>G″ forthe measured frequencies, albeit a clear frequency dependence of G′,indicating relatively long inter-chain crosslink relaxation timesapproaching those in the e-gel.

Silk solutions displayed non-linear shear thinning at lower strainamplitudes followed by shear thickening with increasing shear. ThepH-gels and e-gels showed a large linear viscoelastic regime and asubsequent non-linear regime similar to that observed for silksolutions, including a slight but reproducible strain-stiffening at highshear. Within the measurable strain amplitudes there were no apparentyielding for either pH-gels or e-gels.

The strain hardening was reversible over several cycles. However, when ahigh amplitude shear was applied over several minutes, an irreversibletransition into a stiffer but more brittle gel was observed withapparent yielding. High amplitude shear also led to a slight increase inthe G′ values measured from silk solutions in the linear regime.

The adhesion of silk solution and e-gel on different surfaces wascharacterized using a Dynamic Mechanical Analyzer (DMA) viastrain-controlled, transient tensile testing. Both silk solution ande-gel displayed linear stress-strain behavior for strains up to 20% onstainless steel surfaces. At higher strains, the sample/DMA plateinterface area progressively decreased, which led to significantlydifferent non-linear stress-strain behavior for the silk solution andthe e-gel. For the silk solution, the normal stress peaked at ca. 30 Paat 20% strain and gradually dropped to zero (ca. 150% strain) due tocomplete de-adhesion from the plate. On the other hand, e-gel displayedunique adhesion characteristics when compared to other bioadhesivesystems. After the initial linear regime, the stress progressivelyincreased, while the stress-strain curve showed sporadic fluctuationspresumably due to an interplay between decreasing e-gel/plate interfacearea due to partial de-adhesion and apparent stiffening of e-gel due todehydration and elongational forces. Strains up to 2,500% were recordeduntil failure upon complete de-adhesion. The work of adhesion onstainless steel calculated from the area under the force-displacementcurve for fully hydrated e-gels was 1.09±0.26 mJ (mean±std. dev., n=3).Similar work of adhesion values (1-1.5 mJ) were measured on othersurfaces such as acrylic and wood surfaces, highlighting the versatilityof e-gel adhesion.

Example 13. Use of Silk Films for Electrophysiological Studies and IonChannel Targeted Drug Delivery to Brain Astroglial Cells

Astroglial cell survival and ion channel function are exemplarymolecular targets in terms of neural cell interactions with biomaterialsand/or electronic interfaces. Reactive astrogliosis is the most typicalin vivo response that occurs after brain implants and needs to becontrolled in biomaterial engineering. This cellular phenomena isgenerally characterized by activation of the proliferative state andalteration of expression patterns of astroglial potassium (K+) channels.

Silk interactions with cultured neocortical astroglial cells wereassessed. Cell viability revealed that cell survival was comparable forastrocytes plated on silk-coated glass coverslips compared to thoseplated on poly-D-lysine (PDL), a well-known polyionic substrate, used topromote astroglial cell adhesion to glass surfaces. Comparative analysesof whole-cell single-cell patch-clamp experiments indicated that silkand PDL-coated cells displayed depolarized resting membrane potentials(˜−40 mV), very high input resistance and low specific conductance, withvalues similar to those of undifferentiated glial cells as previouslyreported. Analyses of K+ conductance indicated that silk-astrocytesexpressed large outwardly delayed rectifying K+ current (KDR).

It was next sought to determine whether silk embedded with guanosine(GUO) enabled the direct modulation of astroglial K+ conductance invitro. The results indicated that astrocytes plated on GUO-embedded silkwere more hyperpolarized and expressed inward rectifying K+ conductance(Kir). Collectively the results indicate that silk is a suitablebiomaterial as a platform for studies of astroglial ion channelresponses and related physiology.

REFERENCES

-   1. Andersen R A, Hwang E J, Mulliken G H. Cognitive Neural    Prosthetics. Annu Rev Psychol. 2010; 61:169-C3.-   2. Venkatraman S, Elkabany K, Long J D, Yimin Yao, Carmena J M. A    System for Neural Recording and Closed-Loop Intracortical    Microstimulation in Awake Rodents. IEEE Transactions on Biomedical    Engineering. 2009 January; 56(1):15-22.-   3. Fayad G, Elmiyeh B. Cochlear Implant [Internet]. In: Hakim N S,    editor. Artificial Organs. London: Springer London; 2009 [cited 2011    Dec. 11]. p. 133-6. Available from:    http://www.springerlink.com.ezproxy.library.tufts.edu/content/n318236921756523/-   4. Subbaroyan J, Martin D C, Kipke D R. A finite-element model of    the mechanical effects of implantable microelectrodes in the    cerebral cortex. Journal of Neural Engineering. 2005 Dec. 1;    2(4):103-13.-   5. Lee H, Bellamkonda R V, Sun W, Levenston M E. Biomechanical    analysis of silicon microelectrode-induced strain in the brain.    Journal of Neural Engineering. 2005 Dec. 1; 2(4):81-9.-   6. Cullen D K, Simon C M, LaPlaca M C. Strain rate-dependent    induction of reactive astrogliosis and cell death in    three-dimensional neuronal-astrocytic co-cultures. Brain Research.    2007 Jul. 16; 1158(0):103-15.-   7. Thelin J, Jörntell H, Psouni E, Garwicz M, Schouenborg J,    Danielsen N, et al. Implant Size and Fixation Mode Strongly    Influence Tissue Reactions in the CNS. PLoS ONE. 2011 Jan. 26;    6(1):e16267.-   8. Biran R, Martin D C, Tresco P A. The brain tissue response to    implanted silicon microelectrode arrays is increased when the device    is tethered to the skull. J Biomed Mater Res A. 2007 July;    82(1):169-78.-   9. Harris J P, Capadona J R, Miller R H, Healy B C, Shanmuganathan    K, Rowan S J, et al. Mechanically adaptive intracortical implants    improve the proximity of neuronal cell bodies. Journal of Neural    Engineering. 2011 Oct. 1; 8:066011.-   10. Rousche P J, Pellinen D S, Pivin D P, Williams J C, Vetter R J,    Kipke D R. Flexible polyimide-based intracortical electrode arrays    with bioactive capability. Biomedical Engineering, IEEE Transactions    on. 2001; 48(3):361-71.-   11. Mercanzini A, Cheung K, Buhl D L, Boers M, Maillard A, Colin P,    et al. Demonstration of cortical recording using novel flexible    polymer neural probes. Sensors and Actuators A: Physical. 2008 May    2; 143(1):90-6.-   12. Hess A E, Capadona J R, Shanmuganathan K, Hsu L, Rowan S J,    Weder C, et al. Development of a stimuli-responsive polymer    nanocomposite toward biologically optimized, MEMS-based neural    probes. J. Micromech. Microeng. 2011 May; 21(5):054009.-   13. Kato Y, Saito I, Hoshino T, Suzuki T, Mabuchi K. Preliminary    Study of Multichannel Flexible Neural Probes Coated with Hybrid    Biodegradable Polymer. In: Engineering in Medicine and Biology    Society, 2006. EMBS '06. 28th Annual International Conference of the    IEEE. 2006. p. 660-3.-   14. Takeuchi S, Ziegler D, Yoshida Y, Mabuchi K, Suzuki T. Parylene    flexible neural probes integrated with microfluidic channels. Lab    Chip. 2005 May; 5(5):519-23.-   15. Suzuki T, Mabuchi K, Takeuchi S. A 3D flexible parylene probe    array for multichannel neural recording. In: Neural    Engineering, 2003. Conference Proceedings. First International IEEE    EMBS Conference on. 2003. p. 154-6.-   16. Wester B A, Lee R H, LaPlaca M C. Development and    characterization of in vivo flexible electrodes compatible with    large tissue displacements. J. Neural Eng. 2009 April; 6(2):024002.-   17. Seymour J P, Kipke D R. Neural probe design for reduced tissue    encapsulation in CNS. Biomaterials. 2007 September; 28(25):3594-607.-   18. Chorover S L, Deluca A-M. A sweet new multiple electrode for    chronic single unit recording in moving animals. Physiology &    Behavior. 1972 October; 9(4):671-4.-   19. Lind G, Linsmeier C E, Thelin J, Schouenborg J.    Gelatine-embedded electrodes—a novel biocompatible vehicle allowing    implantation of highly flexible microelectrodes. J Neural Eng. 2010    August; 7(4):046005.-   20. Stice P, Gilletti A, Panitch A, Muthuswamy J. Thin    microelectrodes reduce GFAP expression in the implant site in rodent    somatosensory cortex. Journal of Neural Engineering. 2007 Jun. 1;    4(2):42-53.-   21. Lewitus D, Smith K L, Shain W, Kohn J. Ultrafast resorbing    polymers for use as carriers for cortical neural probes. Acta    Biomaterialia. 2011 June; 7(6):2483-91.-   22. Harris J P, Hess A E, Rowan S J, Weder C, Zorman C A, Tyler D J,    et al. In vivo deployment of mechanically adaptive nanocomposites    for intracortical microelectrodes. J. Neural Eng. 2011 August;    8(4):046010.-   23. Zhong Y, McConnell G C, Ross J D, DeWeerth S P, Bellamkonda R V.    A Novel Dexamethasone-releasing, Anti-inflammatory Coating for    Neural Implants. In: 2nd International IEEE EMBS Conference on    Neural Engineering, 2005. Conference Proceedings. IEEE; 2005. p.    522-5.-   24. Zhong Y, Bellamkonda R V. Dexamethasone-coated neural probes    elicit attenuated inflammatory response and neuronal loss compared    to uncoated neural probes. Brain Research. 2007 May 7;    1148(0):15-27.-   25. Kim D-H, Martin D C. Sustained release of dexamethasone from    hydrophilic matrices using PLGA nanoparticles for neural drug    delivery. Biomaterials. 2006 May; 27(15):3031-7.-   26. Mercanzini A, Reddy S, Velluto D, Colin P, Maillard A, Bensadoun    J-C, et al. Controlled Release Drug Coatings on Flexible Neural    Probes. In: 29th Annual International Conference of the IEEE    Engineering in Medicine and Biology Society, 2007. EMBS 2007.    IEEE; 2007. p. 6612-5.-   27. Zhong Y, Bellamkonda R V. Controlled release of    anti-inflammatory agent a-MSH from neural implants. Journal of    Controlled Release. 2005 Sep. 2; 106(3):309-18.-   28. Purcell E K, Thompson D E, Ludwig K A, Kipke D R. Flavopiridol    reduces the impedance of neural prostheses in vivo without affecting    recording quality. Journal of Neuroscience Methods. 2009 Oct. 15;    183(2):149-57.-   29. Azemi E, Lagenaur C F, Cui X T. The surface immobilization of    the neural adhesion molecule L1 on neural probes and its effect on    neuronal density and gliosis at the probe/tissue interface.    Biomaterials. 2011 January; 32(3):681-92.-   30. David S, Lacroix S. Molecular Approaches to Spinal Cord Repair.    Annual Review of Neuroscience. 2003 March; 26(1):411-40.-   31. Altman G H, Diaz F, Jakuba C, Calabro T, Horan R L, Chen J, et    al. Silk-based biomaterials. Biomaterials. 2003 February;    24(3):401-16.-   32. Meinel L, Hofmann S, Karageorgiou V, Kirker-Head C, McCool J,    Gronowicz G, et al. The inflammatory responses to silk films in    vitro and in vivo. Biomaterials. 2005 January; 26(2):147-55.-   33. Hu X, Shmelev K, Sun L, Gil E-S, Park S-H, Cebe P, et al.    Regulation of Silk Material Structure by Temperature-Controlled    Water Vapor Annealing. Biomacromolecules. 2011; 12(5):1686-96.-   34. Rockwood D N, Preda R C, Yucel T, Wang X, Lovett M L, Kaplan    D L. Materials fabrication from Bombyx mori silk fibroin. Nat.    Protocols. 2011; 6(10):1612-31.-   35. Lin Y-C, Ramadan M, Hronik-Tupaj M, Kaplan D L, Philips B J,    Sivak W, et al. Spatially Controlled Delivery of Neurotrophic    Factors in Silk Fibroin-Based Nerve Conduits for Peripheral Nerve    Repair. Annals of Plastic Surgery. 2011 August; 67(2):147-55.-   36. Huang W, Begum R, Barber T, Ibba V, Tee N C H, Hussain M, et al.    Regenerative potential of silk conduits in repair of peripheral    nerve injury in adult rats. Biomaterials. 2012 January; 33(1):59-71.-   37. Feng Zhang, Rong Liu, Zuo B Q, Qin J Z. Electrospun Silk Fibroin    Nanofiber Tubes for Peripheral Nerve Regeneration. In: 2010 4th    International Conference on Bioinformatics and Biomedical    Engineering (iCBBE). IEEE; 2010. p. 1-4.-   38. Yang Y, Yuan X, Ding F, Yao D, Gu Y, Liu J, et al. Repair of Rat    Sciatic Nerve Gap by a Silk Fibroin-Based Scaffold Added with Bone    Marrow Mesenchymal Stem Cells. Tissue Engineering Part A. 2011    September; 17(17-18):2231-44.-   39. Madduri S, Papaloizos M, Gander B. Trophically and    topographically functionalized silk fibroin nerve conduits for    guided peripheral nerve regeneration. Biomaterials. 2010 March;    31(8):2323-34.-   40. Ghaznavi A M, Kokai L E, Lovett M L, Kaplan D L, Marra K G. Silk    Fibroin Conduits. Annals of Plastic Surgery. 2011 March;    66(3):273-9.-   41. Benfenati V, Toffanin S, Capelli R, Camassa L M A, Ferroni S,    Kaplan D L, et al. A silk platform that enables electrophysiology    and targeted drug delivery in brain astroglial cells. Biomaterials.    2010 November; 31(31):7883-91.-   42. Wittmer C R, Claudepierre T, Reber M, Wiedemann P, Garlick J A,    Kaplan D, et al. Multifunctionalized Electrospun Silk Fibers Promote    Axon Regeneration in the Central Nervous System. Advanced Functional    Materials. 2011 Nov. 22; 21(22):4232-42.-   43. Szybala C, Pritchard E M, Lusardi T A, Li T, Wilz A, Kaplan D L,    et al. Antiepileptic effects of silk-polymer based adenosine release    in kindled rats. Experimental Neurology. 2009 September;    219(1):126-35.-   44. Kim D-H, Viventi J, Amsden J J, Xiao J, Vigeland L, Kim Y-S, et    al. Dissolvable films of silk fibroin for ultrathin conformal    bio-integrated electronics. Nat Mater. 2010 June; 9(6):511-7.-   45. Xia Y, Whitesides G M. Soft Lithography. Angewandte Chemie    International Edition. 1998 Mar. 16; 37(5):550-75.-   46. Hoffmann R, Stieglitz T, Hosseini N H, Kisban S, Paul O,    Ruther P. Comparative Study on the Insertion Behavior of Cerebral    Microprobes. In: Engineering in Medicine and Biology Society, 2007.    EMBS 2007. 29th Annual International Conference of the    IEEE. 2007. p. 4711-4.-   47. Polikov V S, Block M L, Fellous J-M, Hong J-S, Reichert W M. In    vitro model of glial scarring around neuroelectrodes chronically    implanted in the CNS. Biomaterials. 2006 November; 27(31):5368-76.-   48. Polikov V S, Su E C, Ball M A, Hong J-S, Reichert W M. Control    protocol for robust in vitro glial scar formation around microwires:    Essential roles of bFGF and serum in gliosis. Journal of    Neuroscience Methods. 2009 Jul. 30; 181(2):170-7.-   49. Szarowski D H, Andersen M D, Retterer S, Spence A J, Isaacson M,    Craighead H G, et al. Brain responses to micro-machined silicon    devices. Brain Research. 2003 Sep. 5; 983(1-2):23-35.-   50. Skousen J L, Merriam S M E, Srivannavit O, Perlin G, Wise K D,    Tresco P A. Reducing surface area while maintaining implant    penetrating profile lowers the brain foreign body response to    chronically implanted planar silicon microelectrode arrays. Prog.    Brain Res. 2011; 194:167-80.-   51. Jiang C, Wang X, Gunawidjaja R, Lin Y-H, Gupta M K, Kaplan D L,    et al. Mechanical Properties of Robust Ultrathin Silk Fibroin Films.    Advanced Functional Materials. 2007 Sep. 3; 17(13):2229-37.-   52. Lawrence B D, Wharram S, Kluge J A, Leisk G G, Omenetto F G,    Rosenblatt M I, et al. Effect of Hydration on Silk Film Material    Properties. Macromolecular Bioscience. 2010 Apr. 8; 10(4):393-403.-   53. Bjornsson C S, Oh S J, Al-Kofahi Y A, Lim Y J, Smith K L, Turner    J N, et al. Effects of insertion conditions on tissue strain and    vascular damage during neuroprosthetic device insertion. J. Neural    Eng. 2006 September; 3(3):196-207.-   54. A. K. H. Achyuta, V. S. Polikov, A. J. White, H. G. P. Lewis,    and S. K. Murthy, “Biocompatibility assessment of insulating    silicone polymer coatings using an in vitro glial scar assay,”    Macromolecular Bioscience, vol. 10, no. 8, pp. 872-880, August 2010.

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

1-47. (canceled)
 48. An implantable device comprising: a coatedelectrode comprising an electrode substrate and a silk fibroin coating,wherein the electrode substrate has a first stiffness when uncoated,wherein the coated electrode has a second stiffness prior to insertioninto soft tissue, wherein the coated electrode has a third stiffnessafter a predetermined length of time following insertion into softtissue, wherein the second stiffness is greater than the firststiffness, wherein the second stiffness is greater than the thirdstiffness, and wherein the second stiffness is sufficient to penetratesoft tissue.
 49. The implantable device of claim 48, wherein the coatedelectrode has a diameter of less than 2 mm.
 50. The implantable deviceof claim 48, wherein the coated electrode has a Young's modulus of morethan 1 MPa prior to insertion into soft tissue.
 51. The implantabledevice of claim 48, wherein the predetermined length of time is at least1 minute.
 52. The implantable device of claim 48, wherein theimplantable device is a neuroprosthetic device.
 53. The implantabledevice of claim 52, wherein the neuroprosthetic device includes a brainpenetrating electrode, a shunt, or a nerve guide.
 54. A methodcomprising inserting the coated electrode of the implantable device ofclaim 48 into soft tissue of a subject.
 55. An implantable devicecomprising: a coated electrode comprising an electrode substrate and asilk fibroin coating, wherein the coated electrode has a higherstiffness when the silk fibroin coating is in a dry state than when thesilk fibroin coating is in a hydrated state.
 56. The implantable deviceof claim 55, wherein the silk fibroin coating is doped with a conductivematerial.
 57. The implantable device of claim 55, wherein the coatedelectrode has a Young's modulus of more than 1 MPa in the dry state andan elastic modulus or shear modulus of less than 500 kPa in the hydratedstate.
 58. The implantable device of claim 55, wherein the silk fibroincoating comprises an active agent.
 59. The implantable device of claim55, wherein the implantable device is a neuroprosthetic device.
 60. Theimplantable device of claim 59, wherein the neuroprosthetic deviceincludes a brain penetrating electrode, a shunt, or a nerve guide.
 61. Amethod comprising inserting the coated electrode of the implantabledevice of claim 55 into soft tissue of a subject.
 62. An implantabledevice comprising: a coated electrode comprising an electrode substrateand a silk fibroin coating, wherein the coated electrode has a Young'smodulus of at least 1 MPa when the silk fibroin coating is in a drystate, and wherein the coated electrode becomes compliant uponpenetration into soft tissue.
 63. The implantable device of claim 62,wherein the coated electrode has an elastic modulus or shear modulus ofless than 500 kPa upon penetration into soft tissue.
 64. The implantabledevice of claim 62, wherein the coated electrode has an elastic modulusreduced by at least 2-fold upon insertion into soft tissue.
 65. Theimplantable device of claim 62, wherein the implantable device is aneuroprosthetic device.
 66. The implantable device of claim 65, whereinthe neuroprosthetic device includes a brain penetrating electrode, ashunt, or a nerve guide.
 67. A method comprising inserting the coatedelectrode of the implantable device of claim 62 into soft tissue of asubject.